**4. Probiotics and immune modulation**

Probiotics are able to interact at different levels with the host intestinal ecosystem. They may exert effects in the gut lumen via the release of soluble active compounds (metabolites, enzymes), by co-aggregation with pathogens and by intensive cross-talk with the endogenous microbiota (Ait-Belgnaoui et al., 2006; Boirivant and Strober, 2007; Ewaschuk et al., 2008; Haller et al., 2001). They are also known to interact with the intestinal epithelial barrier and its associated mucus, and to initiate immune signaling (Vesterlund et al., 2006; Ohland and MacNaughton, 2010). Specific probiotics are able to exert an effect beyond the gut, influencing the systemic immune system as well as other cell and organ systems, such as liver and brain. For example, certain strains were shown to interact with the enteric nervous system and as such to trigger the gut-brain axis (Cryan and O'Mahony, 2011; Duncker et al., 2008).

Even though we are far from having identified all active compounds that may mediate these interactions, it has been undoubtedly established that bacterial cell surface associated molecules are recognized by the gut immune system. The cell wall of gram-positive bacteria – to which most probiotic bacteria belong – differs from that of gram-negative bacteria by a higher content in peptidoglycan, by the absence of lipopolysaccharides (LPS) and presence of a variety of lipoteichoic acids (LTA) or wall teichoic acids (WTA) instead. In both grampositive and gram-negative bacteria the cell surface may also be decorated by exopolysaccharides (EPS) and/or glycosylated proteins. Altogether these cell surface

Probiotics and Atopic Dermatitis 329

demonstrated to participate to the anti-inflammatory properties of the strain and to impact

While providing an exhaustive review of this area is beyond the scope of the chapter, it might be concluded that the use of a variety of *in vitro* and *in vivo* immune models linked to a good knowledge of bacterial physiology and genetics allowed progress in understanding the mechanisms of action of specific probiotic strains and identification of certain effector molecules. Nevertheless, efforts should be continued in this area while it will also be

Atopic dermatitis or atopic eczema (AD/AE) is a chronic inflammatory disease of the skin, which usually occurs in the early years of life. The skin in AD is extremely dry and itchy and is inflamed - this leads to the characteristic redness, swelling, and scaling pattern often seen in the face and at flexural surfaces of the extremities of patients suffering from AD. AD has been divided in at least 2 different forms: (i) IgE associated or extrinsic dermatitis; (ii) non-

Based on epidemiological and immunological studies, the natural history of AD seems to follow three phases: an initial non-atopic (non-IgE associated) form of eczema occurring in the early infancy followed in 60 to 80% of the cases by a sensitization to food and /or environmental allergens with the development of associated IgE (true AD). Finally, an IgE sensitization to self-proteins is observed in a high proportion of children and adults with

necessary to fill the major gap that remains between preclinical and clinical research.

on intestinal ion channels (Heuvelin et al., 2010).

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Fig. 1. AD classification (Bieber, 2008)

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AD is often the first disorder to manifest in the relay of allergies, usually referred to as the Atopic March (Hahn and Bacharier, 2005; Illi et al., 2004; Spergel and Paller, 2003; Zheng et al., 2011). The prevalence of the disorder has increased dramatically in the last two decades similar to other allergies such as allergic rhinitis and asthma. AD affects mostly infants and young children and according to recent epidemiological studies, 5-20% of

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**5. Atopic dermatitis (AD): Physiopathology of AD** 

components correspond to Microbial Associated Molecular Patterns or MAMPs (also named PAMPS for pathogenic microbes) that may differ substantially from one strain to another. MAMPS are known to bind to specific receptors, the pattern recognition receptors or PRRs, which are expressed by many immune cells and tissues such as the gut epithelium. The binding of MAMPs to PRRs explains how probiotic, commensal or pathogenic bacteria can elicit innate and adaptive immune responses in the host by triggering signaling cascades that in turn lead to the production of cytokines, chemokines and other innate effectors (Abreu, 2010; Kawai and Akira, 2010; Wells et al., 2010). The PRRs belong to three major families: the Toll-like receptors (TLRs), retinoic acid inducible gene I (RIG-I)-like receptors and nucleotide oligomerization domain-like (NOD) receptors (Kawai and Akira, 2010). The TLR and NOD receptors have been shown to play a role in immune activation by probiotics and commensals and so to influence skewing of naïve T cells, regulation of regulatory T cells (Tregs) and activation of antigen presenting cells (APCs) such as dendritic cells (DCs) and macrophages. Activated DCs produce different cytokines in response to different bacterial stimuli and this has consequences for the induction of different T cell subtypes (Baba et al., 2008; Mohamadzadeh et al., 2005). In this sense it is not surprising that different candidate probiotic strains exhibit different immune modulation specificities as they may carry varying MAMPs (Wells, 2011).

Several studies have demonstrated that *in vitro* cytokine profiles elicited from PBMCs, DCs or macrophages vary substantially depending on the species but importantly also on the strain (Wells et al., 2011). Typically, Meijerink *et al.* recently compared the DC response to stimulation by 42 *Lactobacillus plantarum* strains and amounts of IL-10 and IL-12 levels showed up to 39 and 600 fold differences, respectively (Meijerink et al., 2010). This indicates that multiple factors play a role in determining the immune phenotype of a strain. Using genome comparison of *L. plantarum* strains and gene deletion techniques, bacterial genetic loci involved in these specific immune properties could be identified (Marco et al., 2009; Meijerink et al., 2010).

The impact of the LTA has also been highlighted by genetic studies dealing with different *Lactobacillus* species, for example the composition of LTA in *L. plantarum* NCIMB8826 and *Lactobacillus rhamnosus* GG (Grangette et al., 2005; Perea et al., 2007), and presence/absence of LTA in *Lactobacillus acidophilus* NCK56 (Mohamadzadeh et al., 2011) was shown to influence the pro- or anti-inflammatory properties of the wild type and mutant strains. These are examples of studies that combined bacterial physiology and genetics with *in vitro* immune assays in order to generate a hypothesis that could be tested in animal models. They illustrate a nowadays quite active and rapidly evolving field of research. Recent reviews have captured the information that has been gathered on how probiotics can signal through TLR2, TLR2/6, TLR4, TLR9, NOD 1 & 2, and other signaling pathways (Lebeer et al., 2010; Wells et al., 2011).

Different lactobacilli and bifidobacteria have been reported to enhance or restore the barrier function (Resta-Lenert and Barrett, 2003; Ulluwishewa et al., 2011). *In vivo* data support these observations, which were recently reinforced in a human study showing that tight junction proteins of the gut epithelium (biopsies) were regulated upon perfusion of *L. plantarum* WCFS1 in the duodenum of healthy volunteers (Karczewski et al., 2010).

Even though a lot of research has been dedicated to identify which cell surface molecules are operational in the probiotic-host interaction, other molecules such as DNA (Rachmilewitz et al., 2002; Rachmilewitz et al., 2004), metabolites or secreted soluble factors have also been established to play a key role. For example, soluble factors of *Bifidobacterium breve* C50 were

components correspond to Microbial Associated Molecular Patterns or MAMPs (also named PAMPS for pathogenic microbes) that may differ substantially from one strain to another. MAMPS are known to bind to specific receptors, the pattern recognition receptors or PRRs, which are expressed by many immune cells and tissues such as the gut epithelium. The binding of MAMPs to PRRs explains how probiotic, commensal or pathogenic bacteria can elicit innate and adaptive immune responses in the host by triggering signaling cascades that in turn lead to the production of cytokines, chemokines and other innate effectors (Abreu, 2010; Kawai and Akira, 2010; Wells et al., 2010). The PRRs belong to three major families: the Toll-like receptors (TLRs), retinoic acid inducible gene I (RIG-I)-like receptors and nucleotide oligomerization domain-like (NOD) receptors (Kawai and Akira, 2010). The TLR and NOD receptors have been shown to play a role in immune activation by probiotics and commensals and so to influence skewing of naïve T cells, regulation of regulatory T cells (Tregs) and activation of antigen presenting cells (APCs) such as dendritic cells (DCs) and macrophages. Activated DCs produce different cytokines in response to different bacterial stimuli and this has consequences for the induction of different T cell subtypes (Baba et al., 2008; Mohamadzadeh et al., 2005). In this sense it is not surprising that different candidate probiotic strains exhibit different immune modulation specificities as they may

Several studies have demonstrated that *in vitro* cytokine profiles elicited from PBMCs, DCs or macrophages vary substantially depending on the species but importantly also on the strain (Wells et al., 2011). Typically, Meijerink *et al.* recently compared the DC response to stimulation by 42 *Lactobacillus plantarum* strains and amounts of IL-10 and IL-12 levels showed up to 39 and 600 fold differences, respectively (Meijerink et al., 2010). This indicates that multiple factors play a role in determining the immune phenotype of a strain. Using genome comparison of *L. plantarum* strains and gene deletion techniques, bacterial genetic loci involved in these specific immune properties could be identified (Marco et al., 2009;

The impact of the LTA has also been highlighted by genetic studies dealing with different *Lactobacillus* species, for example the composition of LTA in *L. plantarum* NCIMB8826 and *Lactobacillus rhamnosus* GG (Grangette et al., 2005; Perea et al., 2007), and presence/absence of LTA in *Lactobacillus acidophilus* NCK56 (Mohamadzadeh et al., 2011) was shown to influence the pro- or anti-inflammatory properties of the wild type and mutant strains. These are examples of studies that combined bacterial physiology and genetics with *in vitro* immune assays in order to generate a hypothesis that could be tested in animal models. They illustrate a nowadays quite active and rapidly evolving field of research. Recent reviews have captured the information that has been gathered on how probiotics can signal through TLR2, TLR2/6, TLR4, TLR9, NOD 1 & 2, and other signaling pathways (Lebeer et

Different lactobacilli and bifidobacteria have been reported to enhance or restore the barrier function (Resta-Lenert and Barrett, 2003; Ulluwishewa et al., 2011). *In vivo* data support these observations, which were recently reinforced in a human study showing that tight junction proteins of the gut epithelium (biopsies) were regulated upon perfusion of *L.* 

Even though a lot of research has been dedicated to identify which cell surface molecules are operational in the probiotic-host interaction, other molecules such as DNA (Rachmilewitz et al., 2002; Rachmilewitz et al., 2004), metabolites or secreted soluble factors have also been established to play a key role. For example, soluble factors of *Bifidobacterium breve* C50 were

*plantarum* WCFS1 in the duodenum of healthy volunteers (Karczewski et al., 2010).

carry varying MAMPs (Wells, 2011).

Meijerink et al., 2010).

al., 2010; Wells et al., 2011).

demonstrated to participate to the anti-inflammatory properties of the strain and to impact on intestinal ion channels (Heuvelin et al., 2010).

While providing an exhaustive review of this area is beyond the scope of the chapter, it might be concluded that the use of a variety of *in vitro* and *in vivo* immune models linked to a good knowledge of bacterial physiology and genetics allowed progress in understanding the mechanisms of action of specific probiotic strains and identification of certain effector molecules. Nevertheless, efforts should be continued in this area while it will also be necessary to fill the major gap that remains between preclinical and clinical research.
