**2. Human gut microbiota**

The adult human gastro-intestinal tract (GIT) houses about 1014 microbial cells, that outnumber by a factor of 10 the number of cells that compose the human body. This complex microbiota contains over 1000 bacterial types whose number and composition vary along the GIT as a consequence of the different biochemical conditions in the intestine, nutrient availability, age and health status of the host. Of note, the corresponding pool of genes (microbiome) is 150 times larger than the human genome (Eckburg et al., 2005). For this reason, the gut microbiota is sometimes referred to as an organ by itself. It is well established today that this complex microbial community plays an essential role in health and well being. Research conducted with germ-free or gnotobiotic (*i.e.* germ-free animals that were colonized by known bacteria) rodents has unambiguously demonstrated that even if germ free animals are viable when housed in specific conditions and fed with a very nutritious diet, the gut microbiota plays a critical role for normal growth and development (Kelly et al., 2007; Sjogren et al., 2009).

The GIT of mammals is sterile at birth but it becomes rapidly colonized by maternal and environmental bacteria during the delivery. The successive installation of bacterial species

Probiotics and Atopic Dermatitis 327

varies along the gastro-intestinal ecosystem and lactobacilli are typically dominant members of the human vaginal microbiota where they play a key protective role against infections

The health benefits addressed by probiotic research are ranging from their anti-microbial properties to the impact probiotics may have on the host immune system and gut barrier, and on the metabolism and composition of the endogenous microbiota, and as a consequence on the host physiology. The applications are nowadays extending to extraintestinal sites such as the skin, the oral cavity, the urogenital tract and importantly the gutbrain axis. Targeted diseases include bacterial and viral infections, chronic immune disorders such as allergy, inflammatory bowel disease and autoimmune diseases, irritable bowel syndrome, energy and weight management, mood disorders, stress and even psychological disorders such as autism (Asahara et al., 2001; Bin-Nun et al., 2005; Brenner et al., 2009; Carroll et al., 2007; Chapat et al., 2004; Cryan and O'Mahony, 2011; Dani et al., 2002; Iovieno et al., 2008; Madsen et al., 2001; Zareie et al., 2006). From an experimental point of view, the major challenge is to select the most appropriate candidate probiotic strain(s) to reach the selected aim. Therefore, a series of *in vitro* and *ex vivo* assays are used before testing a limited number of strains in animal models mimicking the human disease. Even if time consuming this preclinical research is aiming at facilitating the clinical studies that are mandatory to support the health claim that may be attributed to a probiotic strain. In the past several selection criteria have been applied such as origin of the strain, acid and bile resistance, adhesion to epithelial cells *etc.*, which today rather serve for characterization of candidate probiotic strains (Mercenier et al., 2008). Nowadays, safety and stability of the strains are considered as the main criteria that may lead to exclusion of strains for further applications (Delgado et al., 2008; Grimoud et al., 2010; Gueimonde and Salminen, 2006;

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;

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

(Falagas et al., 2007).

Kalliomaki et al., 2010; Niers et al., 2007).

Duncker et al., 2008).

**4. Probiotics and immune modulation** 

has been well studied (Fanaro et al., 2003; Salminen and Gueimonde, 2005) and was shown to be influenced by several factors such as the mode of delivery and the neonate's diet (Laubereau et al., 2004; Martindale et al., 2005; Negele et al., 2004; Sicherer and Burks, 2008; Zutavern et al., 2006). The next critical phase of bacterial colonization occurs around weaning, when new foods are progressively introduced in the infant's diet. A more complex microbiota is then established that evolves towards the typical adult one over the following years. The progressive bacterial population of the infant intestines has been shown to be of prime importance in the development of a mature gut ecosystem and a functional mucosal immune system. The GIT corresponds to a huge mucosal surface (close to 200 m2 in the adult) that is constantly challenged by external factors such as food components, microbes, toxic compounds, chemicals *etc*. and as such is one of the principal point of entry of pathogens. The GIT thus developed into a sophisticated organ that is able to discriminate between harmful and harmless agents, with an immune system that rapidly mounts defensive responses against infectious microbes while being able to tolerate food or self antigens. This exquisite level of regulation is progressively established as a result of bacterial stimulus including gut microbial colonization in early stages of life. The "hygiene hypothesis" postulates that the lack of adequate microbial challenge linked to modern living conditions in westernized countries is at the origin of the increasing prevalence of chronic immune dysfunctions such as inflammatory bowel disease and allergy (Ege et al., 2008; Schaub et al., 2006; Waser et al., 2007). This postulate is the rationale behind the use of probiotic microorganisms to promote, restore or maintain a healthy status in humans and animals.

### **3. Probiotics and health related benefits**

Probiotics have been defined by a FAO/WHO expert group as « live microorganisms which when administered in adequate amounts confer a health benefit on the host » (2001). This is the most widely accepted definition even if others - that are relatively similar- can be found in literature. This definition implies that the microorganisms are not restricted to bacteria, and yeasts such as *Saccharomyces boulardii/cerevisiae* for example have been studied for their health promoting properties. Typically *S. boulardii* probiotic preparations are commercialized as over the counter (OTC) products to fight diarrhea. Yet, most of the probiotic research has been conducted with lactobacilli and bifidobacteria, even though one strain of *Escherichia coli*, *E. coli Nissle* 1917, and a few strains of *Enterococcus* and *Bacillus* have also been well studied during the last decades. Today, the search for new health promoting microorganisms is extending beyond bacteria isolated from humans to strains originating from fermented food and feed products such as *Pediococcus, Propionobacterium* and *Lactococcus spp*. The bacterial genera *Lactobacillus*, *Enterococcus, Pedicoccus, Propionibacterium, Streptococcus* and *Lactococcus* belong to the family of lactic acid bacteria (LAB) which - as indicated by their name - are able to rapidly transform fermentable carbohydrates in substantial amounts of lactic acid, eventually accompanied by acetic and propionic acid, and butyrate. The capacity of lowering the environmental pH and thus creating an unfavorable milieu for specific pathogens is at the origin of the use of LAB for the preparation and preservation of fermented food and feed products since time immemorial. As a consequence, several LAB species have a "generally-recognised-as-safe" (GRAS) status in the food industry due to their long history of safe human consumption. Several species are also part on the endogenous mammal microbiota and can naturally be found in the oral, intestinal and urogenital tracts of healthy individuals. The number of specific genera of LAB

has been well studied (Fanaro et al., 2003; Salminen and Gueimonde, 2005) and was shown to be influenced by several factors such as the mode of delivery and the neonate's diet (Laubereau et al., 2004; Martindale et al., 2005; Negele et al., 2004; Sicherer and Burks, 2008; Zutavern et al., 2006). The next critical phase of bacterial colonization occurs around weaning, when new foods are progressively introduced in the infant's diet. A more complex microbiota is then established that evolves towards the typical adult one over the following years. The progressive bacterial population of the infant intestines has been shown to be of prime importance in the development of a mature gut ecosystem and a functional mucosal immune system. The GIT corresponds to a huge mucosal surface (close to 200 m2 in the adult) that is constantly challenged by external factors such as food components, microbes, toxic compounds, chemicals *etc*. and as such is one of the principal point of entry of pathogens. The GIT thus developed into a sophisticated organ that is able to discriminate between harmful and harmless agents, with an immune system that rapidly mounts defensive responses against infectious microbes while being able to tolerate food or self antigens. This exquisite level of regulation is progressively established as a result of bacterial stimulus including gut microbial colonization in early stages of life. The "hygiene hypothesis" postulates that the lack of adequate microbial challenge linked to modern living conditions in westernized countries is at the origin of the increasing prevalence of chronic immune dysfunctions such as inflammatory bowel disease and allergy (Ege et al., 2008; Schaub et al., 2006; Waser et al., 2007). This postulate is the rationale behind the use of probiotic microorganisms to promote, restore or maintain a healthy status in humans and animals.

Probiotics have been defined by a FAO/WHO expert group as « live microorganisms which when administered in adequate amounts confer a health benefit on the host » (2001). This is the most widely accepted definition even if others - that are relatively similar- can be found in literature. This definition implies that the microorganisms are not restricted to bacteria, and yeasts such as *Saccharomyces boulardii/cerevisiae* for example have been studied for their health promoting properties. Typically *S. boulardii* probiotic preparations are commercialized as over the counter (OTC) products to fight diarrhea. Yet, most of the probiotic research has been conducted with lactobacilli and bifidobacteria, even though one strain of *Escherichia coli*, *E. coli Nissle* 1917, and a few strains of *Enterococcus* and *Bacillus* have also been well studied during the last decades. Today, the search for new health promoting microorganisms is extending beyond bacteria isolated from humans to strains originating from fermented food and feed products such as *Pediococcus, Propionobacterium* and *Lactococcus spp*. The bacterial genera *Lactobacillus*, *Enterococcus, Pedicoccus, Propionibacterium, Streptococcus* and *Lactococcus* belong to the family of lactic acid bacteria (LAB) which - as indicated by their name - are able to rapidly transform fermentable carbohydrates in substantial amounts of lactic acid, eventually accompanied by acetic and propionic acid, and butyrate. The capacity of lowering the environmental pH and thus creating an unfavorable milieu for specific pathogens is at the origin of the use of LAB for the preparation and preservation of fermented food and feed products since time immemorial. As a consequence, several LAB species have a "generally-recognised-as-safe" (GRAS) status in the food industry due to their long history of safe human consumption. Several species are also part on the endogenous mammal microbiota and can naturally be found in the oral, intestinal and urogenital tracts of healthy individuals. The number of specific genera of LAB

**3. Probiotics and health related benefits** 

varies along the gastro-intestinal ecosystem and lactobacilli are typically dominant members of the human vaginal microbiota where they play a key protective role against infections (Falagas et al., 2007).

The health benefits addressed by probiotic research are ranging from their anti-microbial properties to the impact probiotics may have on the host immune system and gut barrier, and on the metabolism and composition of the endogenous microbiota, and as a consequence on the host physiology. The applications are nowadays extending to extraintestinal sites such as the skin, the oral cavity, the urogenital tract and importantly the gutbrain axis. Targeted diseases include bacterial and viral infections, chronic immune disorders such as allergy, inflammatory bowel disease and autoimmune diseases, irritable bowel syndrome, energy and weight management, mood disorders, stress and even psychological disorders such as autism (Asahara et al., 2001; Bin-Nun et al., 2005; Brenner et al., 2009; Carroll et al., 2007; Chapat et al., 2004; Cryan and O'Mahony, 2011; Dani et al., 2002; Iovieno et al., 2008; Madsen et al., 2001; Zareie et al., 2006). From an experimental point of view, the major challenge is to select the most appropriate candidate probiotic strain(s) to reach the selected aim. Therefore, a series of *in vitro* and *ex vivo* assays are used before testing a limited number of strains in animal models mimicking the human disease. Even if time consuming this preclinical research is aiming at facilitating the clinical studies that are mandatory to support the health claim that may be attributed to a probiotic strain. In the past several selection criteria have been applied such as origin of the strain, acid and bile resistance, adhesion to epithelial cells *etc.*, which today rather serve for characterization of candidate probiotic strains (Mercenier et al., 2008). Nowadays, safety and stability of the strains are considered as the main criteria that may lead to exclusion of strains for further applications (Delgado et al., 2008; Grimoud et al., 2010; Gueimonde and Salminen, 2006; Kalliomaki et al., 2010; Niers et al., 2007).
