The potential role of some phytochemicals in recognition of mitochondrial damage-associated molecular patterns

    The potential role of some phytochemicals in recognition of mitochondrial damage-associated molecular patterns Malgorzata Pierzchalska, Maja Grabacka PII: DOI: Reference:

S1567-7249(16)30050-2 doi: 10.1016/j.mito.2016.06.001 MITOCH 1091

To appear in:

Mitochondrion

Received date: Revised date: Accepted date:

24 February 2016 5 June 2016 7 June 2016

Please cite this article as: Pierzchalska, Malgorzata, Grabacka, Maja, The potential role of some phytochemicals in recognition of mitochondrial damage-associated molecular patterns, Mitochondrion (2016), doi: 10.1016/j.mito.2016.06.001

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ACCEPTED MANUSCRIPT The potential role of some phytochemicals in recognition of mitochondrial damageassociated molecular patterns Malgorzata Pierzchalska*, Maja Grabacka

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Department of Food Biotechnology, Faculty of Food Technology, The University of Agriculture in Kraków, Poland

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*Correspondence to: Malgorzata Pierzchalska Ph.D., Department of Food Biotechnology, Faculty of Food Technology, The University of Agriculture in Kraków, Balicka 122, 30-149, Kraków, Poland; e-mail: [email protected], phone: +48126624796

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Abstract Mitochondria are the source of damage associated molecular patterns (DAMPs). DAMPs modulate responses to stress and trauma in animals, influencing the onset of many diseases. Dietary phytochemicals are potential modulators of immunological status with various cellular targets. In this review the existence of the possible impact of some plant-derived compounds with proven anti-cancer and anti-inflammatory properties (isothiocyanates and curcumin) on DAMPs recognition is highlighted. Special consideration is given to the mtDNA recognizing Toll-like receptor 9 and formyl peptide receptors. In the context of the phytochemicals action, the role of these receptors in epithelial homeostasis is also discussed.

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Keywords: DAMPs; sulforaphane; curcumin; Toll-like receptor 9; formyl peptide receptors, mucosal epithelia, innate immunity Abbreviations: ARE, antioxidant response elements; CMN, curcumin; DAMP, damage associated molecular patterns; DAG, diacyloglycerol; fMLF, formylated chemotactic peptide ̶ f-MetLeuPhe; FPR, formyl peptide receptor; FPRL, FPR-like protein; GSH, glutathione; HMGB1, high mobility group box 1; HSP70, heat shock protein 70; IP3, inositol 1,4,5-trisphosphate; ITC, isothiocyanate; KEAP1, Kelch like Ech-assosiated protein 1; LPS, lipopolysaccharide; mtDNA, mitochondrial DNA; mtDAMPs, mitochondria-derived damage associated molecular patterns; MTC, mitocryptides; NME, N-terminal methionine excision; Nrf2, nuclear factor E2-related factor 2; Pam3Cys, (S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH, trihydrochloride; PDF, protein deformylase; PRR, pattern recognition receptor; ROS, reactive oxygen species; SFN, sulforaphane; SIRS, systemic inflammatory response syndrome; TIR, Toll/interleukin-1 receptor domain; TLR, Toll-like receptor.

Contents 1. Introduction 2. Mitochondria as the source of DAMPs 3. Receptors for mitochondrial DAMPs 3.1. Receptors binding mitochondrial peptides 3.2. Receptor binding mitochondrial DNA 4. Dietary agents potentially influencing mtDAMP signaling 4.1. Isotiocyanates 4.2. Curcumin 5. The possible action of phytochemicals on the mtDAMP recognizing receptors in mucosal epithelia 5.1. Intestinal epithelium 5.2. Airway epithelium 6. Concluding remarks 7. Acknowledgments 8. References 1

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Introduction

Recently, functional food has been gaining a growing interest from both ordinary

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consumers and nutritional and pharmaceutical industry. The recognition of the fact that food

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ingredients, especially of plant origin, are able to positively affect health has a long tradition

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in human culture. Only in recent decades, however, the idea that some phytochemicals modulate inflammation has been put forward (Veldhoen and Veiga-Fernandes, 2015). Numerous latest studies conducted in vitro or in vivo and some clinical trials link the

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immunological status to dietary phytochemicals (Tab. 1). At the same time, claims that some dietary agents can effectively improve immunity evoke serious doubts and suspicions (Jantan et al., 2015). Such skepticism can be only addressed by carefully designed research based on

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the vigorously expanding knowledge of the biological processes on the cellular level. Along with some membrane receptors and cytoplasmic enzymes of numerous cell signaling pathways, mitochondria were also identified as a possible molecular target of many

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phytochemicals. Signals generated in mitochondria are partially responsible for protective or

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therapeutic potentials of many plant-derived compounds (for review see Grabacka et al., 2014).

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The “danger theory” of immunity proposed in early 90’s states that immunological system of vertebrates is more focused on recognition of so called “danger signals” (involve both fragments of invading pathogens and parts of damaged cells) than on distinguishing

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between self and nonself-antigens (Matzinger, 1994). This hypothesis found the confirmation in the recent discoveries of broad range of endogenous molecules forming damage associated molecular patterns (DAMPs). Particularly important group of DAMPs comprise fragments of broken mitochondria, that leaking from damaged cells or generated actively by stressed tissues are able to trigger local and systematic immunological responses acting through the same patterns recognition receptors (PRR) that recognize pathogens (Jeong and Lee, 2011). In this review we would like to discuss the possibility that the immunological response evoked by mitochondrial generated DAMPs (mtDAMPs) is influenced by phytochemicals. To debate this hypothesis one has to realize what kind of “danger signals” are produce by mitochondria and what type of receptors are capable of binding mtDAMPs. We will focus on the details of these ligands-receptors interactions to analyze them as potential targets of some bioactive food-derived compounds.

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2.

Mitochondria as the source of DAMPs

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Undamaged cell keep all its content behind the solid “fence” of the cell membrane and

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only small proportion of proteins produced by cellular machinery have the signal allowing their exocytosis and only such proteins are bound to be exported into neighboring tissue or to

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the bloodstream. In a living organism, cells that have become redundant are extricated by programmed cell death and during this processes cells building blocks are degraded in the control way and eliminated by tissue macrophages (apoptosis), or alternatively dispersed

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locally and into the bloodstream (necroptosis). Any cell component performing its normal functions in a living healthy cell might be suspected to act as a DAMP, when accidentally or

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purposely liberated. Indeed, fifteen years ago one of the constituents of necrotic cells, the cytoplasmic chaperon HSP60, was shown to activate cytokines synthesis acting through Tolllike receptor 4 (TLR4), well known before as the bacterial lipopolysaccharide (LPS) receptor

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(Ohashi et al., 2000). Many other abundant cytoplasmic components with various structure,

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like ATP, F-actin, uric acid crystals or single stranded RNA, were also identified as DAMPs. The nuclear components are equally able to activate immune system, among them DNA

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chaperone, high mobility group box 1 (HMGB1), is the best studied example. Moreover, HMGB1 is recognized not only by TLR2 and TLR4, but also by advanced glycation endproduct receptor (RAGE) (Vénéreau et al., 2015). Fragments of mitochondria are especially prone to evoke “danger signal” as they are

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structurally related to their prokaryotic ancestors and a lot of PRRs recognize molecular patterns of bacterial characteristic. Damaged mitochondria were shown to be a potent stimulator of neutrophils in vitro (Hazeldine et al., 2015). It was shown that injection of mitochondrial debris into the circulation of mouse can cause a systematic inflammatory response syndrome (Zhang et al., 2010) and that mtDAMPs in the circulation are responsible for frequent pneumonia cases in traumatic patients (Li et al., 2016). Both mitochondrial DNA with unmethylated CpG motifs and formyl peptides produced by mitochondrial translation machinery were identified as the endogenous ligands of Toll-like receptor 9 (TLR9) and formyl peptide receptor (FMR), respectively. As mitochondrial proteins and DNA are constantly produced by these organelles, it is necessary to protect cells against accidental activation by their own components. Therefore, the signal evoked by mitochondrial DAMPs is finely tuned by various mechanisms, particularly by the regulation of the receptor compartmentalization. The perturbations in signaling from mtDAMPs are linked to many 3

ACCEPTED MANUSCRIPT pathological conditions in animals and humans. The elevated level of circulating mtDNA was observed in patients after serious traumatic injury and recognized as a reason of systemic inflammatory response syndrome (SIRS) that strongly resembles sepsis (Simmon et al.,

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2013). The mtDNA is suggested to play a role in pathogenesis of organ failure and in the

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onset of many chronic autoimmune diseases, e.g., rheumatoid arthritis (Collins and Wilson, 2014), diabetes (Gülden and Wen, 2014) and systematic lupus erythromatosus (Lood et al.,

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2016). Noteworthy, the blood level of mtDNA is elevated not only in diabetics (Czajka et al., 2015), cancer (Schwarzenbach et al., 2011) and hypertension (McCarthy et al., 2014) patients, but also is significantly increased in HIV carriers (Cossarizza et al., 2011) and in some

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generally healthy elderly subjects (Pinti et al., 2014). The presence of this DAMP and activation of TLR9 is among the major causes of “inflamm-ageing” – the status of chronic

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inflammation characteristic of old organisms, as well as it contributes to endothelial dysfunction in pre-diabetic states and diabetes (Alvarado-Vásquez, 2015).

Receptors for mitochondrial DAMPs

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3.

Various receptors present in numerous types of both immunocompetent and other cells

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in the body orchestrate the response to DAMPs. From this plethora of receptors only those that bind mitochondrial peptides and mtDNA will be described below. 3.1.

Receptors binding mitochondrial peptides

Newly synthesized proteins in bacteria are N-terminally formylated, because the

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bacterial initiator tRNA (tRNAf) carries N-formylated methionine and translation starts from the formyl-methionine incorporation instead of regular methionine, as is in eukaryotes. Formylated peptides are formed also in the mitochondria, as these organelles retained the translation machinery of their prokaryotic predecessors. Short synthetic peptides with Nterminal formylated methionine, such as fMetLeuPhe (fMLF), were shown to be extremely strong chemotactic agents for neutrophils and monocytes, exerting the biological activity even at the concentrations below 10-11 M (Schiffmann et al., 1975; Showell et al., 1976). This demonstrates the alertness of the innate immunity system towards bacterial pathogens. Numerous mitochondrial proteins such as mitochondrially encoded respiratory complexes subunits (cytochrome b, cytochrome c oxidase, NADH dehydrogenase, ATP synthase) possess N-terminal formyl methionine residues. These proteins or their cleavage products, but not mitochondrial nonformylated proteins (encoded in nucleus and imported from cytoplasm), are able to induce chemotaxis of granulocytes (Carp, 1982). In bacteria, as well as in plastids 4

ACCEPTED MANUSCRIPT and mitochondria, there is an N-terminal methionine excision (NME) machinery that deformylates some proteins. In mammalian mitochondria NME is achieved through removal of the formyl group from N-terminal methionine by protein deformylase (PDF) and this step

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enables cutting off the methionine by methionine aminopeptidase (MAP1D). PDF activity

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seems to be necessary for the proper functioning of mitochondria, because its inhibition decreases the mitochondrial protein translation and impairs assembly of respiratory

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complexes, which affects ATP synthesis in human cells (Escobar-Alvarez et al., 2010). Nevertheless, the detailed mass spectrometry studies of the bovine mitochondrial proteome revealed that only one of the thirteen mtDNA encoded proteins is posttranslationally

methionine residues (Walker et al., 2009)

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deformylated (subunit III of cytochrome c oxidase), whereas the rest retains formyl-

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The host response to the formylated peptides of endogenous or exogenous origin is carried out through the specific receptors: FPR and two homologous FPR-like proteins, FPRL1 and FPRL2, which share 69% and 56% of amino acid sequence identity with FPR

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(Prossnitz and Ye, 1997). All three receptors are expressed predominantly in the

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immunocompetent cells (monocytes, polymorphonuclear leukocytes and dendritic cells) but they are also present in hepatocytes, glia and endothelium, as well as intestinal, lung, thyroid

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and adrenal epithelia. Formyl peptide receptors cooperate with Gi protein and belong to metabotropic G-protein coupled receptors with seven transmembrane domains (Prevete et al., 2015). In leukocytes, the receptors reside in the cell membrane, but are also present in the granules and secretory vesicles (Sengeløv et al., 1994). The signal transduction from these

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receptors involve a phospholipase C activation and release of secondary signal transmitters, such as diacyloglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 induces a rapid mobilization of intracellular calcium stores that leads to switching on chemotaxis, enhanced superoxide production, degranulation and secretion of lysosomal enzymes (Partida-Sánchez et al., 2001; Showell et al., 1976; Wilkinson, 1979). FPR receptor was the first identified receptor responsible for chemotactic response of neutrophils towards formylated bacterial peptides, such as fMLF (Showell et al., 1976; Boulay et al., 1990) and is often called a high affinity receptor for these ligands. FPRL1 and FPRL2 need several orders of magnitude higher concentrations of ligands, which mostly comprise formylated or nonformylated proteins of endogenous and pathogenic origin, or their cleavage products. The importance of FPR in effective and rapid anti-microbial response is illustrated by the data obtained from the experiments on FPR-/- mice, whose neutrophils failed to react to fMLF in vitro, leading to

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ACCEPTED MANUSCRIPT increased lethality in the animals challenged with Listeria monocytogenes, in comparison to the wild type littermates (Gao et al., 1999). Recently, endogenous biologically active peptides, so called mitocryptides (MCTs)

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have been described (Marutani et al., 2015; Mukai et al., 2008). MCTs are fragments of the

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mitochondrial proteins (products of either mitochondrial or nuclear genes), such as cytochrome c oxidase, cytochromes or other components of respiratory chain that are formed

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in damaged tissues, e.g., ischemic heart (Marutani et al., 2015). Their function is not entirely clear, but they are believed to act as mtDAMPs and promote phagocytosis and other innate immune protection mechanisms during cellular or tissue injury, but also may take part in

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sterile inflammation development. A cryptide MCT-2 seems to be particularly interesting, because it is a selective FPRL1 agonist and does not activate FPR (Seki et al., 2011). MCT-2

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is a fragment of mitochondrial cytochrome b containing N-terminal formyl-methionine residue and 14 other amino acids (Mukai et al., 2009). An interesting property of MCTs is their ability to trigger an “accumulative signaling”, i.e. the mixture of various cryptides in low

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concentrations induces biological response, such as phagocytosis, whereas the same

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concentrations of the peptides administered separately do not exert any effect (Marutani et al., 2015). This may suggest that there is a certain kind of cooperation in the receptor-ligand

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interaction that enhances the response when the stimulus exceeds a threshold concentration. 3.2. Receptor binding mitochondrial DNA For many years it has been believed that mtDNA cannot be methylated due to the lack of histone backbone and the doubtful possibility of methyltransferases transport into It

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mitochondria.

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that

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methyltransferase 1 (DNMT1) could be located in mitochondria and human and murine mtDNA is to some extent methylated (Iacobazzi et al., 2013; Ghosh et al., 2014). The pattern of this epigenetic modification is, however, different from that observed in nuclear DNA, as the methyl groups are mainly attached to non-symmetrical cytosine residues (Bellizzi et al., 2013). It was also shown that mtDNA contains numerous cytosine-phosphate-guanine palindromic sequences (so called CpG islands) in which – in contrast to majority of such structure in genomic DNA – cytosine is not methylated. Such unmethylated motifs, resembling those of bacterial origin, can be considered a typical DAMP. In mammals they are recognized by TLR9 and are able to modify the inflammatory response (Fang et al., 2010; Ries et al., 2013). TLR9 is a member of the group of microbial recognition receptors homologous to Drosophila Toll protein. The toll protein was first discovered and characterized as essential receptor in fly embryonic development (Hoshimoto 1988) and 6

ACCEPTED MANUSCRIPT subsequently proved to be crucial for the innate immunity, conservative and present in many members of animal kingdom. TLR9 is a type I integral membrane protein. The N-terminal domain containing 25 leucine rich repeats (LRR) is flanked by two cysteine-rich capping

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regions. The disulfide bounds formed inside these regions are believed to be important for

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effective ligand recognition and signal transduction. The signaling domains of TLRs are known as Toll/interleukin-1 receptor (TIR) domains because of their homology to the

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signaling domains of IL-1R family members (Carpenter and O’Neill, 2009). The C-terminal cytoplasmic TIR-like domain is critical for receptor function as it attracts adaptor molecules (Fekonja et al., 2012). Principally, the dimerization of TLRs happens after ligand binding and

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promotes recruitment of adaptor molecules to intracellular TIR domain. Nevertheless, TLR9 activation is unique because its dimer is formed in ER independently to the process of ligand binding (Peter et al., 2009). Preformed dimers are then transported from ER to endosomes

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where they undergo proteolysis – the N-terminal part of ectodomain is cut off by proteases (Park et al., 2009). This processing affects the ability of ligand binding and subsequent signal

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transduction events (Ewald et al., 2008). Of note, TLR9, which is present not only in

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mammals but also in amphibians and fish, is absent in birds. As it was shown in chicken, avian cells recognize unmethylated DNA using another receptor called chTLR21, which

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shares the ligand specificity, but not the structural features with its mammalian analog (Keestra et al., 2010; Brownlie et al., 2009). TLR9 is expressed both in the immune cells, like lymphocytes, eosinophils (Kvarnhammar and Cardell, 2012), neutrophils, monocytes, macrophages and dendritic cells

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(Fiola et al., 2010; Dickie et al., 2010) and in various other cell types, such as cardiac fibroblasts (Ohm et al., 2014), cardiomyocytes and neurons (Shintani et al., 2013), endothelial cells (Li et al., 2004) and in airway (Zhang et al., 2011; Schneberger et al., 2009), intestinal (Pedersen et al., 2005) and other organs epithelial cells and cancers. In search for molecular background of pathological states and physiological phenomena there have been attempts to expand our knowledge of TLR9 role by analyzing its gene polymorphism in human population and by studying the phenotype of TLR9 knockout mice. Several human TLR9 gene single nucleotide polymorphisms (SNPs) both in the protein coding and the promotor region have been identified as affecting receptor function or expression. Some of them are linked to the pathophysiology or susceptibility of various chronic diseases of both infectious and autoimmunological background. The examples of such disorders associated to TLR9 variants are tuberculosis (Schurz et al., 2015), Helicobacter pylori induced gastritis ((Ng et al., 2010), symptomatic malaria (Omar et al., 2012) and 7

ACCEPTED MANUSCRIPT inflammatory bowel disease (Shang et al., 2016). The insight to TLR9 pleiotropic activity was gained through the observation that TLR9 knockout mice were more prone than wild type littermates to develop insulin resistance (when fed on high-fat diet) or necrotizing colitis

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(Gribar et al., 2009). They are also characterized by shorter survival in case of Salmonella

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enterica Serovar Typhimurium infection (Zhan et al., 2015). The comparison of double knockout mice (TLR9-/-, ApoE-/-) with ApoE-/- mice indicated that TLR9 activation might

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be protective in atherosclerosis (Koulis et al., 2014). TLR9 was of interests also for cancer biologists. Some authors concluded that TLR9 might prevent transformation in the colon (Fűri et al., 2013). TLR9 expression level was checked in colorectal polyps with various

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histological characteristic and was shown to be downregulated in patients, who eventually suffered from malignant colorectal lesions (Eiró et al., 2012). The fact that activation of TLR9

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seems to promote immunological homeostasis both in intestinal and airway epithelium is the basis of using CpG deoxynucleotide as potential therapeutic agents in asthma, chronic obstructive pulmonary disease (COPD) (Aryan et al., 2014), inflammatory bowel diseases

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(IBD) (Abreu, 2010) and ulcerative colitis (Musch et al., 2013). The same group of biodrugs

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was tested as an adjuvant in cancer immunotherapy, but some other reports presenting the cancer cells reaction to TLR9 activation in vitro rose concerns about the safety of such an

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approach (Huang et al., 2008). For example, in prostate cancer cells (Moreira et al., 2015) and glioblastomas (Wang et al., 2010) TLR9 signaling was shown to be crucial for cancer propagation and promotion of more aggressive phenotype. Another caveat was connected with the increased TLR9 expression in non-healing wounds of diabetes patients (Singh et al.,

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2015). The role of TLR9 activation in pathogenesis of autoimmunological diseases, including systematic lupus erythematosus, was also suggested but remains controversial (Celhar and Fairhurst, 2014). All these studies shed light on the safety of TLR9 ligands clinical applications.

4.

Dietary agents potentially influencing mtDAMP signaling

The analysis of contemporary literature reveals that there are few experimental clues or epidemiologic evidences concerning the influence of dietary agents on mtDAMP binding to their receptors. We postulate that such possible link can be suggested, based on the fact that the effect of some bioactive compounds present in food on signaling from other structurally related peptides has been already demonstrated (Tab. 2). From known phytochemicals of the suggested anti-inflammatory potential two groups are particularly important from the point of 8

ACCEPTED MANUSCRIPT view of recognition of signals generated by mitochondria: isothiocyanates and curcumin derivatives.

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4.1. Isotiocyanates

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Isotiocyanates (ITCs) are produced by plants from Brassicaceae family as a part of defense machinery directed against insects, pathogens and herbivorous animals, operating as

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myrosinase-glucosinolate system. In the intact plant tissue myrosinase is spatially separated from its substrates, glucosinolates that reside in the intracellular vacuoles. Upon the plant tissue injury and vacuole breakage the enzyme comes to contact with glucosinolates cleaving

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off the sugar moiety to form active ITC (Angelino et al., 2015). Some ITCs, for example 4methylsulfinylbutyl isothiocyanate (sulforaphane, SFN) from broccoli, are not only natural

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pesticides, but also have been shown to exert profound anti-inflammatory and anti-cancerous activities in animal cells (Gupta 2014). As was suggested in the excellent review by NegretteGuzmán et al., SFN is able to evoke both cytoprotective or pro-apoptotic effects depending on

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the treated cells type (e.g. normal or transformed) and concentration used (1-5 M, in contrast

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to 10-50 M) (Negrette-Guzmán et al., 2013) by modifying different molecular targets (Fig. 1). SFN enters cells by passive diffusion and is immediately conjugated to cytoplasmic

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glutathione (GSH) (Mi et al., 2011b; Shapiro et al., 2001). These two processes lead to a rapid accumulation of ITCs in cells (the concentration in cytoplasm can reach milimolar range). The SFN-GSH conjugates are transported back to the intestinal lumen or to the bloodstream

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by xenobiotics transporters, namely multidrug resistance associated protein-1 and -2, respectively. In consequence, the cells are depleted from reduced glutathione and become vulnerable to negative consequences of mitochondrial reactive oxygen species (ROS) production. What is more, SFN and other ITCs can be attached to specific proteins that expose cysteine residues in the reduced form in the physiological pH (Mi et al., 2011a). ITCs reacts also with and ε-amino-containing lysine or proline residues. Due to these reactivities SFN has many potential molecular targets inside the cell. For example, SFN modifies oxidative stress sensor, Kelch like Ech-assosiated protein 1 (KEAP1). KEAP1 is an actin binding protein, which connects nuclear factor E2-related factor 2 (Nrf2) to Cul3-dependent E3 (Cul3) ubiquitin ligase complex, promoting the nuclear factor ubiquitylation in the cytoplasm and preventing its transfer to the nucleus. When SFN is attached to one of KEAP1 cysteine residues (C151), the sensor is not able to bind Nrf2 that switches the balance from cytoplasmic degradation to migration to the nucleus (Kensler et al., 2013). When in the 9

ACCEPTED MANUSCRIPT nucleus, Nrf2 binds to antioxidant response elements (ARE) in the promotors of many genes active in cellular stress response – so called phase II proteins (Bellezza et al., 2010). The studies on cells lacking Nrf2 indicate its importance for anti-inflammatory activity of ITC

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resulting from interference with both the Nrf2 and NF-κB signaling pathways. Boyanapalli et.

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al compared the effect of phenethyl isothiocyanate (PEITC) and curcumin (CMN) on the LPS stimulated macrophages, isolated from Nrf2 knockout and wild-type mice (Boyanapalli et al.,

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2014). They showed that the inhibitory effect of phytochemicals upon expression of proinflammatory genes, cytokines and proteins, was much more pronounced in wild-type cells. Some of the effects of ITCs were nevertheless still visible in cells lacking the expression

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of Nrf2. Other studies recognize the LPS receptors, TLR4, as a direct target of ITCs. Youn et al. demonstrated employing the mouse monocytic cell line, RAW264.7, that SFN interferes

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directly with TLR4 oligomerization by modifying some cysteines in its extracellular domain (Youn et al., 2010). In contradiction to previously described results, these authors were unable to detect any differences between the ITC-induced inhibition of pro-inflammatory reaction in

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mouse embryonic fibroblasts isolated from Nrf2 KO-mice, as compared to its wild-type

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counterparts. It was also suggested by others that SFN can impaired TLR4 signaling by modifying the 133 cysteine residue of co-receptor MD2 – the protein connecting TLR4 to its

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ligand (Koo et al., 2013). Moreover, Shibata et. al. showed that iberin, structurally closely related to SFN, covalently binds to mouse TLR4 and TLR2 preventing effective signal transduction upon their ligand binding (Shibata et al., 2014). Folkard et.al demonstrated that SFN is covalently linked to cysteine 609 in the extracellular domain of human TLR4 at

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reducing condition and suggested that this explained the suppression of proinflammatory response of THP-1 cells and human peripheral blood mononuclear cells stimulated with LPS in the presence of the compound (Folkard et al., 2014). The ability of cysteines in the ectodomain of TLR9 to form adducts with ITCs was not investigated so far. But the alignment of human TLR4 and TLR9 sequences shows structural similarity and parallel localization in cysteine rich flanking region between cysteine 609 of TLR4 and cysteine 790 of TLR9. If the adduct between cysteine 790 of TLR9 and SFN is indeed formed in living cells, there is a possibility that the signal transduction upon ligand binding might be altered in the similar way to the LPS-TLR4 pathway.

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Fig. 1. The confirmed cellular targets of isothiocyanates, which may take part in the response to mtDAMP. Dietary factors of isothiocyanates structure are known to influence the membrane receptors (by impairing TLR pathway or as membrane anion channels inhibitors). In cytoplasm they could be conjugated to reduced glutathione or cytoplasmic SH-containing proteins (e.g., Keap1). Isothiocyanates were also known as inhibitors of histone deacetylases (Kaufman-Szymczyk et al., 2015) or NFκB (Tortorella et al., 2015), what directly modulate the expression of inflammation-related genes by epigenetic mechanisms.

Others ITCs of plant origin, such as 4'4-diisothiocyanostilbene -2'2'-disulfonic acid

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(DIDS) and 4-amido-4'-isothiocyanostilbene-2'2' disulfonic acid (SITS) have also been

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reported to interfere with the FPR ligand recognition and signaling in the human neutrophils stimulated with formyl peptides (Skubitz et al., 1989). SITS and DIDS were known to be

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specific blockers of membrane chloride channels, but Skubitz and coauthors showed that these isothiocyanates not only blocked the binding of fMLF to FPR, but also reduced the number of the FPR receptors on the neutrophil surface. The consequence of DIDS hampering

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with the FPR ligand recognition, the neutrophil responses to fMLF, such as myeloperoxidase release were blocked by DIDS (Smith et al., 1984). It cannot be excluded that the inhibitory effects of ITCs on FPR signaling involve the modification of the cysteine residues crucial for the G-protein binding and further signal transduction. The Cys124 and Cys126 located in the cytoplasmic edge of the III transmembrane domain and in the III cytoplasmic domain, respectively, are conserved in all the members of FPR family (Miettinen et al., 1999). The substitutions of these cysteine residues to serine lead to the protein G uncoupling and impaired signaling (Miettinen et al., 1999). The importance of Cys126 is illustrated by the existence of the SNP leading to Cys126 to Trp substitution in FPR is observed in patients suffering from local aggressive periodontitis (LAgP) (Jones et al., 2003). One of the clinical traits of LAgP is impaired reaction of neutrophils to bacterial pathogens. The studies performed on Chinese hamster ovary (CHO) cells expressing the mutant FPR revealed the

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ACCEPTED MANUSCRIPT lower affinity of the receptor to fMLF ligand, reduced G-protein coupling and weak chemotaxis towards fMLF (Jones et al., 2003). The detailed analysis of the FPR extracellular ligand binding domain revealed the

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existence of two Cys residues (Cys 98 and Cys 176) that form disulfide bond, which is crucial

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for the binding of the formyl peptide. The exchange of these residues to alanines, that prevent the disulfide bond formation, abolish the ligand binding (Perez et al., 1994). Summing up, it is

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conceivable that the ITCs interfere with formyl peptide recognition through the interaction with the FPR cysteine residues, both in cytoplasmic and extracellular regions. This would suggest that the modulation of the FPR ligand recognition and signaling can be exerted by

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ITC phytochemicals with various abilities to cross the cellular membranes. 4.2. Curcumin

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Curcumin (1,7-bis[4-hydroxy 3-methoxy phenyl]- 1,6-heptadiene-3,5-dione, CMN) is the main active ingredient found in popular spice curry. It is isolated from rhizome of domestic plant Curcuma longa used for centuries in Asian cuisines and traditional medicine.

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Chemically it belongs to polyphenolic compounds known from its health promoting effects

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attributed to their antioxidant properties. Nowadays various biological activities of CMN are carefully studied on the cellular level to confirm not only anti-oxidant but also anti-

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inflammatory, anti-infection and anti-tumor and anti-diabetic properties (Soni and Salh, 2012). The studies that focused also on the attempts to increase of its relatively low bioavailability lead to the discovery and characterization of more potent analogs or alternative ways of distribution of this phytochemicals (Prasad et al., 2014). Administration of CMN in

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vivo and in vitro changes the activity of many signaling pathway including suppressing of NFκB and enhancing Nrf2 activity (Shehzad and Lee, 2013). In case of inhibition of proinflammatory reactions most researchers point at the ability of CMN to block IKK kinase what prevents the downstream events and the transport of NF-κB to the nucleus (Jobin et al., 1999; Pan et al., 2000). Decoté-Ricardo et. al. have shown that CMN inhibits both proliferation and IgM secretion in B cells stimulated by the TLR4 ligand (LPS) and TLR9 ligand (CpG) but not by TLR2 ligand (Pam3Cys), what suggests that it acts upstream of common mediators activated by all these receptors (Decoté-Ricardo et al., 2009). Some authors also suggested that CMN interferes directly with TLR4 receptors dimerization (Youn et al., 2006). Interesting results emerged from the in silico analysis of curcumin interaction with cellular proteins conducted with bioinformatics tools. Using module-based protein interaction network analysis Gan et. al. looked for the main protein targets of CMN and found that TLR9 receptor was a node seed in one of the modules in curcumin-protein network (Gan 12

ACCEPTED MANUSCRIPT et al., 2015). The authors concluded that TLR9 might play a major role in the antiinflammatory action of the “gold spice”. If it is the case, curcumin would influence the onset of diseases in which elevated level of mtDNA, one of the TLR9 ligands, was reported.

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CMN has been shown to blunt the neutrophil pro-inflammatory response not only to

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LPS but also to fMLF by inhibiting NF-B signaling (Antoine et al., 2013). Other authors demonstrated the inhibition of LPS-induced neutrophil invasion in air pouches model in mice,

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as well as the leukocyte chemotaxis towards fMLF in vitro (Antoine et al., 2013; Srivastava, 1989; Uthayashanker and Rita, 2015). These observations suggest some involvement of inhibition of FRP signaling by this dietary agent, although it has not been investigated yet, if

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the FPR is directly a molecular target of CMN.

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The possible action of phytochemicals on the mtDAMP recognizing receptors in mucosal epithelia

Both TLR9 and FPRs are not only present on immune cells but also on actively

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proliferative epithelial cells of various origin. In epithelia, especially those in contact with

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commensal bacterial species in physiological conditions, activation of these receptors is more

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tightly connected to homeostasis than to inflammation (as outlined in the Fig. 2). 5.1 Intestinal epithelium

Intestinal epithelium is built of polarized cells, mainly absorptive enterocytes with microvilli on their apical surface, which are in constant contact with commensal bacteria.

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Although, the intestinal microflora is a rich source of PRR ligands, in healthy gut it does not trigger inflammatory response. The compartmentalization of PRR and their signaling

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machinery is the base of “polarizing-tolerizing” mechanism proposed by Lee et. al. (Lee et al., 2008). Enterocytes form the barrier between external and internal environment. The influence on animal cells exerted by plant secondary metabolites, which, although bioactive, possess no

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nutritional value, is determined by their bioavailability. Being present in the normal diet and

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taken orally with food or as supplements, phytochemicals have to cross the intestinal barrier to reach other cells in the body. The bioactive compounds are subjects of the efficient and

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unspecific detoxification machinery in the cytoplasm of intestinal cells. In this enzymatic process the chemicals are hydrolyzed, oxidized, conjugated to other compounds to increase solubility in water and, eventually, extracted back to the intestinal lumen or to the

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bloodstream (Nasef et al., 2014). The effectiveness of the detoxification depends on the compound structure and leads to a very low bioavailability of some agents (e.g. CMN, resveratrol) (Prasad et al., 2014; Dempe et al., 2013; Juan et al., 2010) or extraordinary efficient transfer through the intestinal epithelium to the bloodstream in the case of others (e.g. SFN) (Atwell et al., 2015). It is obvious that intestinal absorptive enterocytes, which decide the fate of phytochemicals by metabolizing them, are also potential targets of their actions. The influence of a given compound in case of normal epithelium and intestinal derived tumors in situ depends on its concentration in intestinal lumen but not in the blood, so even agents with a low bioavailability are potentially able to affect intestinal functions. FPR2 are selectively expressed in the colonic epithelial cells of the crypt region in human (Babbin et al., 2007) and mice (Chen et al., 2013) and was shown to be located in the cells apical and lateral membranes. It was demonstrated that the activation of FPR1 in the mice colon crypts took place during the contact with bacteria form Lactobacillus sp that 14

ACCEPTED MANUSCRIPT stimulated ROS production. The process was suggested to be responsible for the induction of cell proliferation and, eventually, healing of the wound (Alam et al., 2014). The in vitro study showed also that activation of human FPR1 in intestinal cell line (SK-CO15) enhances cell

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migration and monolayer “wound” closure (Leoni et al., 2013).

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In a healthy epithelium TLR9 compartmentalization has an important role in evoking tissue tolerogenic phenotype (Lee et al., 2008). In phagocytes the receptor is localized mainly

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in endosomal compartment. In the gut epithelium, however, the substantial amount of the receptor molecules are located in the basolateral and apical membrane (Yu et al., 2014). It was postulated that upon being transferred to the cell membrane the receptor is still able to

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bind its ligands, but fails to relocate NF-B to the nucleus. To effectively evoke proinflammatory signals TLR9 has to be directed to the endolysosomal compartment, where

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the dimerized receptor is cleaved by cathepsins and endopeptidases (Yu and Gao, 2015). In line with these hypothesis, TLR9-/- mice were shown to suffer from the delayed healing after chemical gut damage (Rose et al., 2012) and TLR9 synthetic agonists were shown to protect

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from the radiation injury (Saha et al., 2012). There are also some notions about dual role of

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TLR9 activation in inflammation-associated intestinal cancers (Sipos et al., 2014). We cannot exclude the possibility that in such a situation the substances, which prevent the ligands-TLR9

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binding would deepen the homeostatic imbalance in cancer or delay the recovery after an injury. In case of SFN such influence would be, however, counterbalanced by the cytoprotective effect of Nrf2 activation, which is responsible for beneficial effects of Lactobacillus gut colonization during recovery from radiation injury in both flies and mice

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(Jones et al., 2015; Moreira et al., 2015). On the other hand, the expansion of cancer cells which can respond to DNA released from dying neighboring cells is limited due to TLR9 activation and this process could be congested in the presence of some dietary agents (Tuomela et al., 2013). In case of carcinomas originated from the gut epithelium the activation of c-MET (hepatocytes growth factor receptor - HGFR) by HGF (scatter factor) plays a profound role in establishment of invasive phenotype (e.g. enhanced cell migration and matrix remodeling capacity, epithelial-mesenchymal transition) acting through the translocation to nucleus of NF-κB (Hao et al., 2015). Some phytochemicals, isothiocyanatostilbenes and curcumin, are not only found to be inhibitors of c-MET (Gray et al., 2015 ; Hu et al., 2015) but also lowers cancer cell migration rate. Benzyl isothiocyanate (BITC) slows down migration and invasion of human colon cancer HT29 cells by inhibition metalproteinases activity (Lai et al., 2010). Activation of NF-κB was shown also to be crucial for antiproliferative impact of BITC on colon cancer cells, as tested on HT-29 line in vitro (Abe 15

ACCEPTED MANUSCRIPT et al., 2014). It could be assumed that, as HGFR and TLR signaling pathways crossreact at NF-κB the anti-cancerous impact of some phytochemicals may be achieved regardless which

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receptor is a primary molecular target of their action.

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Fig. 2. The putative role of phytochemicals in mtDMAPs recognition by immunocompetent and epithelial cells. The cell damage evokes the leakage of mitochondrial material (DNA – unmethylated CpG and formylated peptides - fPep), which can act, when outside the maternal cell, as DAMPs and bind to appropriate receptors (e.g., TLR9 or FPR1/2) of the healthy cells in the neighborhood or in a distant target tissue in the body. The outcome of such activations (solid arrows) varies depending on the cell type. In immunocompetent cells (e.g., neutrophils) activation leads to the production of proinflammatory cytokines and chemokines; in mucosal epithelia (e.g., intestinal or bronchial cells) the same receptors activation promote homeostasis (proliferation, migration and wound healing). To assess the potential modulatory action (dashed lines) of sulforaphane (SFN) or curcumin (CMN) on the tissue damage (in effect of trauma, radiation, autoimmunological and allergic diseases and cancer) both these response variants should be taken into account.

5.2. Airway epithelium

In cells of other epithelia and mucosa, like skin keratinocytes and airway epithelial cells or tumors derived from these tissues, there is a direct connection between blood level of phytochemicals and their potential impact on cells proliferation or malignant transformation. Some phytochemicals, like curcumin, were also administered by inhalation or intranasally in case of experimental treatment of disease connected to airway epithelial injury (Chauhan et al., 2014). In the airway epithelium, which can be easily damaged by external agents like pollutants or microbes, the presence of various DAMPs is recognized by numerous TLR receptors including TLR9 (Whitsett and Alenghat, 2015). TLR agonists are tested as an adjuvants for immunovaccine, widely applied in treatment of severe allergic diseases, including asthma (Pollinex® Quattro vaccine contains synthetic TLR4 ligand and AIC vaccine comprises 1018 immunostimulatory sequence, a TLR9 synthetic agonist) (Aryan and 16

ACCEPTED MANUSCRIPT Rezaei, 2015). The use of synthetic TLR4 and TLR9 ligands in monotherapy is also currently tested in clinical trial. The CpG based immunotherapy seems to be a promising strategy in allergy and atopic bronchial asthma treatment, as it promotes the switch from Th2 to Th1

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response in the airways. Nevertheless, so far the results of clinical trials conducted with the

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use of various CpG adjuvants are inconclusive, especially in the case of atopic and asthmatic children (Aryan et al., 2014). Some in vitro studies on primary human bronchial epithelial

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cells from asthmatic and nonasthmatic donors suggested that TLR9 ligands can act directly on epithelial cells restoring its barrier function compromised in asthma (Kubo et al., 2015). The presence of TLR9 was also confirmed in the primary nasal epithelial cells in culture (van

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Tongeren et al., 2015) and TLR9 ligands were found to be effective in case of chronic allergic rhinosinusitis (Rhee et al., 2004).

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Surprisingly, dietary agents with the potential to impair the TLR signaling are also tested as add-on therapeutics in allergic diseases. Sulforaphane was recently proved beneficial both in asthma mouse model (Park et al., 2012) and in patients (Brown et al., 2015). In mouse

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model of asthma CMN was proved to be effective in both reducing inflammation (Yuan et al.,

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2014) and bronchial remodeling (Chauhan et al., 2014). The clinical study on effects of CMN in the treatment of asthma was also conducted, but statistically significant improvement was

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not observed (Abidi, 2014). The in vitro experiments showed the pro-apoptotic CMN impact on human bronchial epithelial cells exposed to cigarette smoke extract (CSE). These results indicated the potential of CMN to modulate the regeneration of airway epithelium stimulated

6.

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by endogenous ligands liberated from cells injured by CSE (Liu et al., 2008).

Concluding remarks

The notion that anti-inflammatory food components such as polyphenols or n-3 polyunsaturated fatty acids influence the immune system homeostasis is well accepted. However, the concept that plant derived bioactive compounds can affect the innate immunity, particularly microbial pattern recognition, seems to be much less acknowledged. Here we have presented the possible mechanisms through which phytochemicals may modulate TLR9 and FPR mediated recognition of mitochondrial DAMPs. The importance of such mechanisms

for

physiological

and pathological

conditions

should

be confirmed

experimentally in future research. From the current knowledge we can conclude that the potential influence of ITCs and CMN on mtDAMPs recognition and the blocking of subsequent signaling could act as a double-sword in some pathological situations and such supplements should be carefully investigated prior to the introducing into clinical practice. 17

ACCEPTED MANUSCRIPT Nevertheless, the rapidly expanding awareness of the possible means to modulate innate immunity system may contribute to development of new strategies to treat traumatic stresses,

Acknowledgement

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7.

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inflammatory or autoimmune diseases and cancer.

Authors acknowledge the financial support of Polish National Science Center (grant no.

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2013/09/B/N29/00285 to M.P.)

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ACCEPTED MANUSCRIPT

8.

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reduce colonization and attenuate gastritis in Helicobacter pylori-infected mice and humans. Cancer Prev. Res. 2, 353–360. doi:10.1158/1940-6207.CAPR-08-0192 Youn, H.S., Kim, Y.S., Park, Z.Y., Kim, S.Y., Choi, N.Y., Joung, S.M., Seo, J. a, Lim, K.-M.,

NU

Kwak, M.-K., Hwang, D.H., Lee, J.Y., 2010. Sulforaphane suppresses oligomerization of TLR4 in a thiol-dependent manner. J. Immunol. 184, 411–419.

MA

doi:10.4049/jimmunol.0803988

Youn, H.S., Saitoh, S.I., Miyake, K., Hwang, D.H., 2006. Inhibition of homodimerization of Toll-like receptor 4 by curcumin. Biochem. Pharmacol. 72, 62–69.

D

doi:10.1016/j.bcp.2006.03.022

TE

Yu, S., Gao, N., 2015. Compartmentalizing intestinal epithelial cell toll-like receptors for immune surveillance. Cell. Mol. Life Sci. 72, 3343–3353. doi:10.1007/s00018-015-

CE P

1931-1

Yu, S., Nie, Y., Knowles, B., Sakamori, R., Stypulkowski, E., Patel, C., Das, S., Douard, V., Ferraris, R.P., Bonder, E.M., Goldenring, J.R., Tony Ip, Y., Gao, N., 2014. TLR sorting by Rab11 endosomes maintains intestinal epithelial-microbial homeostasis. EMBO J. 33,

AC

1–14. doi:10.15252/embj.201487888 Yuan, S., Cao, S., Jiang, R., Liu, R., Bai, J., Hou, Q., 2014. FLLL31, a derivative of curcumin, attenuates airway inflammation in a multi-allergen challenged mouse model. Int. Immunopharmacol. 21, 128–136. doi:10.1016/j.intimp.2014.04.020 Zhan, R., Han, Q., Zhang, C., Tian, Z., Zhang, J., 2015. Toll-like receptor 2 (TLR2) and TLR9 play opposing roles in host innate immunity against Salmonella enterica serovar typhimurium infection. Infect. Immun. 83, 1641–1649. doi:10.1128/IAI.02870-14 Zhang, C., Wu, X., Zhao, Y., Deng, Z., Qian, G., 2011. SIGIRR inhibits toll-like receptor 4, 5, 9-mediated immune responses in human airway epithelial cells. Mol. Biol. Rep. 38, 601–609. doi:10.1007/s11033-010-0146-7 Zhang, Q., Raoof, M., Chen, Y., Sumi, Y., Sursal, T., Junger, W., Brohi, K., Itagaki, K., Hauser, C.J., 2010. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature 464, 104–7. doi:10.1038/nature08780 33

ACCEPTED MANUSCRIPT Table 1. Clinical trials (completed or ongoing) testing sulforaphane and curcumin as a treatment for inflammatory disorders Treated disorder

Sulforaphane

Oral (dietary supplement) Oral Topical (gel)

Atopic Asthma

TE

Topical (gel)

D

Oral (dietary supplement) Oral (dietary supplement)

MA

Oral Topical (mouthwash)

CE P

Oral (dietary supplement)

AC

NCT01315665 NCT00894712

SC R

Oral

NU

Oral

Curcumin

Cystic Fibrosis Radiation induced dermatitis in cancer patients Response to live attenuated influenza virus in smokers and nonsmokers Helicobacter pylori induced gastritis H. pylori infection Oral mucositis in patients receiving chemotherapy Atopic Asthma

Reference or clinical trial identification number from www.clinicaltrials.gov NCT01845493

T

Route of application

IP

Phytochemical

Radiation induced dermatitis in cancer patients Radiation induced dermatitis in cancer patients Rheumatoid arthritis

Oral (dietary supplement)

Ulcerative colitis

Oral (dietary supplement) Oral (dietary supplement)

Crohn Disease

Oral (dietary supplement)

Osteoarthritis

Topical (mouthwash) Oral (dietary supplement)

Gingivitis Anterior uveitis

Inflammatory Bowel Disease

34

NCT01269723

Yanaka et al., 2009 Galan et al., 2004 NCT00475683 NCT02300727 Elad et al., 2013 NCT01179256 NCT01246973 NCT01042938 Ryan et al., 2013 NCT02556632

NCT02543931 NCT00752154 Chandran and Goel, 2012 NCT02277223 NCT02683759 NCT00793130 Lang et al., 2015 Singla et al., 2014 NCT02255370 NCT00889161 Suskind et al., 2014 Holt et al., 2005 Pinsornsak and Niempoog, 2012 Kuptniratsaikul et al., 2014 Panahi et al., 2014 Henrotin et al., 2014 Nakagawa et al., 2014 Muglikar et al., 2013 Lal et al., 1999

ACCEPTED MANUSCRIPT Table 2. The in vitro and in silico studies indicating the influence of some anti-inflammatory dietary agents on DAMP recognition Phytochemical

Possible action

References

TLR4

sulforaphane

The inhibition of receptor oligomerization in murine monocytic cell line;

Youn et. al., 2010

T

Receptor

The inhibition of murine LPS-MD2-TLR4 complex formation. The suppression of signaling by covalent modification of cysteine residues 246 and 609 in human TLR4 The inhibition of ligand-dependent TLR4 dimerization The inhibition of TLR2-TLR6 complex formation The inhibition of ligand recognition

TLR2

iberin

Formylpeptides

FPR

SITS, DIDS

curcumin

In silico analysis of curcumin-human proteins interactions points at TLR9 as one of main targets

CE P

TE

D

TLR9

AC

CpG

NU

HSP70

MA

curcumin

SC R

IP

Putative DAMP ligand HSP70, HSP90, HMGB-1

35

Koo et al., 2013 Folkard et al., 2014 Youn et 2006 Shibata et 2016 Skubitz et 1989 Smith et 1984 Gan et 2015

al., al., al., al., al.,

ACCEPTED MANUSCRIPT HIGHLIGHTS

T

IP

SC R

NU MA D TE



CE P



Liberated fragments of mitochondria mediate the systemic response to stress and trauma, modulate the onset and resolution of inflammation, as well as the initiation and progression of cancer. Dietary phytochemicals with anti-inflammatory and anti-cancerous properties have many possible molecular targets in a cell, including membrane receptors and signaling molecules. The activation of receptors recognizing mitochondrial damage-associated molecular patterns and their downstream signaling molecules, could be potentially compromised by isothiocyanates and curcumin. This phenomenon may exert an impact on the onset of many diseases.

AC



36