Initial Probability Model

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Upon a microbial attack, host cells undergo massive changes in their transcriptional program, mobilizing genes involved in key processes (e.g., immunity, cell death/survival, and adhesion/motility) to trigger an appropriate response (Jenner and Young 2005, Bierne et al., 2012). It is thus not surprising that successful pathogens have developed specific mechanisms to deregulate the expression levels and/or kinetics of these defence genes. Host transcription factors are first obvious targets to reprogram the genome and bacteria use diverse tricks to alter their function. For instance, bacterial factors can hijack cellular signaling pathways that activate or sequester transcription factors e.g. nuclear factor kappa B (NF-κB), signal transducer and activator of transcriptions (STATs), or activator protein 1 (AP-1) in the cytosol of targeted cells, or manipulate their half-lives via posttranslational modifications (Ribet and Cossart 2010; Perrett et al. 2011). Some bacteria, such as the phytopathogen Xanthomonas, even produce transcriptional activators that function as eukaryotic transcription factors. However, selective activation or silencing of specific genes not only depends on transcription factors, but also on their cross talk with epigenetic modulators, which regulate DNA accessibility by controlling the chromatin structure. Epigenetic modifications of chromatin during development and in response to distinct environmental factors contribute to adult phenotypic variability and susceptibility to a number of diseases, including cancers and metabolic and autoimmune disorders (Portela and Esteller 2010).

Several studies have shown that certain infectious agents (Helicobacter pylori, Streptococcus bovis, Chlamydia pneumoniae, Campylobacter rectus, Epstein-Barr virus, hepatitis viruses, Human papilloma virus, polyomaviruses, etc.) can contribute to the host epigenetic changes resulting in the onset and progression of some diseases, especially in malignancies (Gilbert et al., 2010). The importance of DNA methylation events associated with bacterial infections is also becoming increasingly appreciated. The best documented example is H. pylori infection that induces aberrant DNA methylation in the human gastric mucosa, strikingly at promoters of genes found methylated in gastric cancer cells (Ushijima and Hattori 2012). H. pylori-associated hypermethylation occurs, for instance, at the E-cadherin gene 1 (CDH1), tumor-suppressor genes e.g., Upstream stimulatory factor 1/2 USF1/2 (Yan et al. 2011), as well as to CpG islands of miRNA genes (Ando et al. 2009).

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Figure 2.13. Environmental factors capable of inducing immune complexes and subsequent NF-kB activation and Translocation (Hoesel and Schmid, 2013)

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The ability of H. pylori to induce DNA methylation in gastric mucosa was confirmed in the gerbil animal model and, interestingly, this effect was diminished on treatment with the immunosuppressor cyclosporin A (Niwa et al., 2010). Indeed, in contrast to ethanol or NaCl stimuli that induce neutrophil infiltration in the stomach, H. pylori-mediated inflammation triggered lymphocyte and macrophage infiltration, which appears to have a key role in induction of methylation (Hur et al., 2011). Thus, although the mechanisms by which H. pylori induces DNA hypermethylation are still unclear, the infection-associated inflammatory response is a tempting explanation (Ushijima and Hattori 2012). Among signals resulting from chronic inflammation, elevated levels of IL1β and nitric oxide (NO) are proposed to contribute to influence the recruitment of DNMTs at specific loci. A role of mammalian gut microbiota, as epigenetic modifying factor, in the pathogenesis of metabolic syndrome and associated diseases has been meant. Thus, the different biotic and abiotic signals can produce changes in gene expression that can persist after the effect has ceased (Oka et al., 2011).

The first step in any epigenetic study is global DNA methylation analyses which allow the detection and identification of DNA methylation. These approaches do not require previous knowledge of the genome of reference, and most rely on a prior enzymatic/chemical hydrolysis of DNA to obtain 2′-deoxymononucleosides, followed by the subsequent separation by chromatographic means such as High Performance Liquid Chromatography (HPLC) (Raid, 2013) or High Performance Capillary Electrophoresis (HPCE) (Raid, 2013), and a final detection step by UV spectroscopy or mass spectrometry. Alternatively, the global content of DNA methylation can also be quantified by enzymatic approaches such as the Luminometric Methylation Assay (LUMA) (Raid, 2013).

Interestingly, bacterial DNA load correlated inversely with advanced disease, a finding that could have broad implications in diagnosis and staging of breast cancer (Xuan et al., 2014).

Previous studies of microbial causes of breast cancer have focused on specific viruses and their potential contributions to breast cancer. While HPV infection has been reported by some groups to be associated with breast cancer (Heng et al., 2009, Xuan et al., 2014), others have failed to find a correlation (Lindel et al., 2007). Similarly, some groups have reported that up to 50% of breast tumors are EBV-positive (Fina et al., 2001, Xuan et al., 2014), while others have been unable to detect the virus in breast tumors altogether (Glasser et al., 1998). In contrast to viruses, bacteria in the breast have been studied to a far lesser extent. Several groups have investigated the bacteria responsible for infections stemming from breast implant procedures using

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based methods (Pittet et al., 2005). Further, the breast milk of healthy women has been shown to harbour an abundance of bacterial species including commonly found skin bacteria (Cabrera-Rubio et al., 2012). Bacteria in the breast have been studied in the context of infections and in healthy individuals, but no comprehensive study of bacteria in breast cancer has been reported.

Microbes inhabiting the human body outnumber human cells 10:1. Their influence on human health and disease is a new and rapidly expanding area of research. Microbes have been linked to diseases as varied as obesity, colon cancer and colitis (Castellarin et al., 2012, Xuan et al., 2014). Examples of few microbe-induced epigenetic dysregulations include:

Campylobacterrectus induces Methylation of Igf2 promoter region to cuase silencing of Igf2 P0 promoter by CpG methylation in the placenta; Helicobacter pylori induces Polycomb-repressive marks pinpoint the promoters to be silenced to enable silencing of selected promoters by CpG methylation; Epstein-Barr causes up-regulation of DNMT1, 3A, 3B via the JNK-AP-1 to induce silencing of E-cadherin promoter; Human adenovirus causes stimulation of E2F activity, up-regulation of DNMT1; association with DNMT1, stimulation of DNMT1 activity to induce dysregulation of DNMT1, 3A, 3B; Human papillomavirus, through association with DNMT1, cause stimulation of DNMT1 activity; increasing histone acetylation. Activation of E2F and CDC25A promoters; Hepatitis B virus induces Up-regulation of DNMT1 via the cyclin D1-CDK4/6-pRb-E2F1 and p38MAPK pathways; up-regulation of DNMT3A1 and DNMT3A2;

Down-regulation of DNMT3B thereby causing silencing of tumor suppressor genes

Microbial manipulation of host epigenetic marks as obligate intracellular parasites has helped develop numerous ways of hijacking cell processes to facilitate the completion of their life cycle and sometimes to evade the immune responses of their host. Microbes that cause persistent infections are likely to benefit from heritable epigenetic changes in host transcription that produce an environment for their latent or persistent state without having to continuously express the initiating effectors (Virgin et al., 2009). Host genes involved in cell cycle progression, senescence, survival, inflammation and immunity are prime candidates as targets for such epigenetic control. Some chronic bacterial infections are also associated with malignancy, the most and widely studied being Helicobacter pylori infection of human gastric mucosa. Moreover, many microbes have evolved ways of eluding the immune response and, again, epigenetic changes in host cells have been implicated in these processes. Viral infection can deregulate patterns of repressive histone modifications that could then precipitate aberrant DNA methylation and the reprogramming of infected cells and their progeny.

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Epigenetic mechanisms regulate expression of the genome to generate various cell types during development or orchestrate cellular responses to external stimuli. Recent studies highlight that bacteria can affect the chromatin structure and transcriptional program of host cells by influencing diverse epigenetic factors (i.e., histone modifications, DNA methylation, chromatin-associated complexes, noncoding RNAs, and RNA splicing factors), Bierne et al., (2012), revealed that the molecular bases of the epigenetic language and then describe the current state of research regarding how bacteria can alter epigenetic marks and machineries. Bacterial-induced epigenetic deregulations may affect host cell function either to promote host defence or to allow pathogen persistence. Thus, pathogenic bacteria can be considered as potential epimutagens able to reshape the epigenome. Their effects might generate specific, long-lasting imprints on host cells, leading to a memory of infection that influences immunity and might be at the origin of unexplained diseases (Bierne et al., 2012).

Various bacterial products can affect them in many ways, through activation of signaling cascades or directly in the nucleus. So far, most of the reported chromatin modifications induced by bacteria are histone acetylation/deacetylation and phosphorylation/dephosphorylation events generated through activation of host cell signalling cascades by bacterial components (e.g., microbe-associated molecular patterns, metabolites, and virulence factors). The effects are complex, because they differ according to the bacterial agonist, cell type, and kinetics parameters. Among the host signaling pathways that a number of bacteria activate, mitogen activation protein kinase (MAPKs) e.g. ERK and p38, NF-κB and Phosphatidylinositol 3-Kinase (PI3K) pathways are known to activate the kinases that phosphorylate H3S10 in the nucleus (i.e., Mitogen and stress-activated protein kinase 1/2 (MSK1/2), Inhibitor of KappaB Kinase alpha IKKα, and Protein kinase B (AKT/PKB), respectively) (Baek 2011). Any bacterial stimulus activating these pathways has therefore the potential to induce H3S10 phosphorylation and associated acetylated histones (Bierne et al., 2012).

The innate immune system senses the invasion of pathogenic microorganisms through the Toll-like receptors (TLR), which recognize specific molecular patterns present in microbial components. Microorganisms (prokaryotes) and tumor cells share a common feature: both contain unmethylated CpG motifs at a higher frequency than eukaryotes and normal cells, respectively. The innate immune system detects unmethylated CpG motifs using TLR-9 (Takeshita et al., 2004). The release of unmethylated CpG DNA during an infection provides a danger signal to the innate immune system, triggering a protective immune response that

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improves the ability of the host to eliminate the pathogen (Klinman, 2004). Activation of the TLR-9 subsequently leads to the activation of a cascade including nuclear factor-κB (NFkB) that culminates in the activation and proliferation of immune cells.