Studies investigating the composition of the intestinal microflora in humans have shown the pivotal role of commensal bacteria in the development of allergies. Studies comparing the microflora composition of atopic and non-atopic infants also show significant differences. Non-atopic infants have more colonization of Bifidobacteria and Lactobacillus in the intestine while there is much colonization of Clostridia in the intestine of atopic infants. However several reports also suggest that there are no significant differences between the two groups (21,22). The exact role of intestinal flora in the development of atopic disease in the childhood is still not clarified (23). In addition to these quantitative differences in the Bifidobacterium, qualitative differences have also been observed. The Bifidobacteria from atopic infants were found to induce much higher levels of pro-inflammatory cytokines in vitro whereas the Bifidobacteria from non-atopic infants induced more secretion of anti-inflammatory cytokines. In addition to different secretion ability, differential adhesion to intestinal mucosa may also result in a different or reduced stimulation of the immune system through the gut-associated lymphoid tissue (GALT) (24).

Obesity is associated with the changes in the relative abundance of the two dominant bacterial divisions, the Bacteroids and the Firmicutes. The relative proportion of Bacteroids is decreased in obese people compared to lean people while the proportion of Firmicutes is increased in obese people. Obese microbiome has an increased capacity to harvest energy from the diet and colonization of germ-free mice with an obese microbiota results in greater increase in total body fat without any increase in food consumption than colonization with a lean microbiota (25-27).

Metagenomic analysis revealed the close relatedness of bacteria with the development of T1D associated autoimmunity in young child. Especially the proportion of Bacteroides ovatus was increased in children with T1D while the proportion of Firmicutes, CO19 was increased in normal healthy children (28).

All these results collectively imply that gut microbiota could be an additional contributing factor to the pathophysiology of obesity, T1D as well as atopic dermatitis, which in turn suggest that bacterial markers can be used for early diagnosis for these disorders. In addition, supplementation of large amount of bacteria that negatively correlated with the disease state may be beneficial for the prevention of these disorders.

Based on the hygiene hypothesis, in addition to environmental factors, the intestinal microflora is another contributor to allergic disease by substantially affecting the mucosal immunity. Allergic responses are thought to arise if there is absence of microbial exposure while the immune system is developing (24,29). The gastrointestinal tract is an important interface between host and environment and has the dual role of facilitating the absorption of nutrients while excluding pathogens. The human intestines are inhabited by at least 400 different bacterial species, with the greatest density in the large intestine, where concentration of 10¹¹-10¹² cells/g of luminal contents can be found (30). Because a large part of the intestinal microflora cannot be cultured with current culture techniques, it has been suggested that the number of microflora species in the human intestine may exceed 1,000 species (24). Although some intestinal bacteria are potential pathogens, the relationship between the intestinal microflora and the human host is mostly symbiotic in healthy individuals.

The largest mass of lymphoid tissue of the human body can be found in the gastrointestinal tract and is called as the gut-associated lymphoid tissue (GALT) which includes Peyer's patch and mesenteric lymph node (MLN) (31). GALT interact with intestinal bacteria which are sampled by dendritic cells (DCs) and intestinal epithelial cells through the pattern recognition receptors such as toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) (32-34). The intestinal antigens also can be captured by DCs in the Peyer's patch through M cells on the surface of enterocytes. DCs in lamina propria can capture antigens directly by dendrites (35-37). Intestinal microflora seems to be involved in the induction of oral tolerance which is established in the periphery to specific antigen after ingestion of orally delivered protein (antigen) and plays a pivotal role in the induction and maintenance of peripheral tolerance mediated by regulatory T cells (38,39).

Most studies delineating the role of resident microorganisms in regulation of immune system and inflammation have focused on the gut microbiome, since gut is the main site of exposure and contains a wide diversity of microbes. However, skin was considered to just have a physical barrier function to protect our body from the attack of pathogens. Interestingly recent studies discovered that skin microbiome also have similar function that of gut microbiome (40). Microbial profiling by metagenomic analysis has revealed the presence of highly diverse commensal communities in skin sites (41). Moreover, cutaneous inflammatory disorders, such as psoriasis and atopic dermatitis have been associated with imbalance of the cutaneous microbiota (42,43). Indeed, microbial products from skin commensals are known to exert immunoregulatory effects (44). Yasmine Belkaid group reported that the roles for the skin microbiota in controlling the local inflammatory milieu. They showed that protective immunity to a cutaneous pathogen is critically dependent on the skin but not on gut microbiota (45). Furthermore, skin commensals activate the function of local T cells through the interleukin-1 receptor (IL-1R)-MyD88 signaling pathway (45). These findings indicate the critical role of the skin residual commensal bacteria not only as a distinctive feature of tissue compartmentalization also as a key regulator of the immune system in health and disease.

The intestinal commensal bacteria play diverse functions in regulating nutrient metabolism including absorption of indigestible carbohydrate and production of short chain fatty acid, amino acid and vitamin. Microflora provides additional energy by digesting exogenous and endogenous substrates, such as fibers and mucins. Microflora also provide a protective barrier against incoming bacteria (46). This protection is mediated by several different mechanisms including competition for nutrients and binding sites and production of antimicrobial substances (37,47).

Probiotics could stimulate the immune system by modulating the composition and/or activity of the intestinal microbiota (48,49). The immunomodulatory role of probiotics has been shown in diverse types of inflammatory immune disorders both, in gastrointestinal diseases and in non-gastrointestinal diseases. The beneficial effect of probiotics is strain-specific and their effector mechanisms are also quite diverse. Some probiotics generate IL-10 producing Tr1 or CD4⁺Foxp3⁺ Treg cells (50), while others could enhance immunity by producing a large amount of IL-1β, IL-12 or TNF-α (51). For example Lactobacillus reuteri induces the production of IL-12 and TNF-α from DCs while Lactobacillus casei inhibits the production of pro-inflammatory cytokines by producing anti-inflammatory cytokines such as IL-10. Therefore it is crucial to use specific probiotics or their mixture to target specific immune disorders. Most of the allergic diseases are associated with a shift of the Th1/Th2 balance toward a Th2 response, which induces secretion of Th2 type cytokines such as IL-4, IL-5 and IL-13 leading to higher production of IgE. Recently, we have demonstrated that IRT5 probiotics, a mixture of 5 probiotics, could suppress diverse immune disorders through the generation of CD4⁺Foxp3⁺ Tregs (52). Oral administration of IRT5 probiotics generated rDCs (iDO^high, Cox2^high, IL10⁺, TGFβ⁺), which can convert effector CD4⁺Foxp3^- T cells into CD4⁺Foxp3⁺ regulatory T cells in the mucosal immune system. In addition, oral administration of the IRT5 probiotics suppressed ongoing experimental AD and pathogenesis of experimental arthritis as well (52). Currently we are investigating to identify potent probiotic strains that could induce the production of immune-modulatory cytokines such as IL-10 and TGF-β or enhancing Th1 response to improves the symptoms of allergic disease (37,53).

DCs function as a professional antigen presenting cells (APC) and have the ability to activated T cells and directly regulate Th1 and Th2 cell. Especially DCs lead to the induction of regulatory T cells through the secretion of IL-10 and TGF-β and help to regulate immune response by the production of IL-10 from Tr1 cells and TGF-β from Th3 cells. Once DCs capture antigens in the presence of proper inflammatory stimuli, they migrate to secondary lymphoid organs and simultaneously undergo a maturation process. During this maturation, DCs produce diverse cytokines and express surface molecules such as MHC, CD40, CD83, CD80 (B7.1) and CD81 (B7.2). Recently several reports suggest that probiotics can induce the maturation and cytokine secretion by DCs. Different probiotics exert differential effect on the cytokine profiling by DCs such as IL-12, TNF-α and IL-10 (54-56). Recently we have demonstrated that administration of IRT5 probiotics mixture increases the generation of CD4⁺Foxp3⁺ Tregs in the mesenteric lymph nodes (MLN) through the generation of CD11c⁺ regulatory dendritic cells (rDCs) that highly express high levels of IL-10, TGF-β and indoleamine 2,3-dioxygenase (iDO) (52), which in turn potentiated the generation of CD4⁺Foxp3⁺ Tregs from the pool of CD4⁺CD25^- cells. These results suggest that probiotics or a mixture of them that can induce regulatory DCs in the MLN, which in turn induce the production of CD4⁺Foxp3⁺ cells, could represent an attractive potential therapy for the treatment of diverse inflammatory immune disorders.

Atopic dermatitis (AD) is caused by diverse pathogenic factors including genetic susceptibility, environment trigger, skin barrier dysfunction, bacterial infection and immune dysregulation (5). AD is a complex immune reaction mediated by both Th1 and Th2 immune responses (6). Th2 type cytokines including IL-4, IL-5 and IL-13 play important role in the development of AD by increasing the levels of serum IgE and blood eosinophils in AD patients (57). In the later stage of AD where infection mediated inflammation occurs, Th1 type cytokines such as IFN-γ and IL-12 mediate chronic stage of atopic dermatitis (5,58,59). IFN-γ is also involved in the maintenance of chronic stage of atopic dermatitis by elevating the expression of CCL17 (TARC) and CCL22 (MDC) that are involved in the recruitment of effector T cells to the inflamed site (60). IFN-γ increases the sensitivity of Fas-mediated apoptosis of keratinocytes, which is considered to be a key pathogenic event in eczematous dermatitis (61). Based on many clinical as well as animal studies, therapeutic or preventive effects of probiotics on allergic disease have been intensively investigated as summarized hereinafter.

Several randomized controlled trials (RCT) have investigated the effect of probiotics on the prevention of atopic dermatitis. In these trials probiotics were given to infants with a high risk of developing allergy, starting immediately after birth and mothers also received probiotics during the last week of pregnancy. The first trial reported that a 50% reduction of the incidence of AD in the probiotics group compare to placebo group at the age of 2 year and this effect was maintained until at 7 years (62). A clinical trial reported that administration of probiotics only reduced the incidence of IgE-associated AD without altering other allergic diseases (63). In another report, consumption of Lactobacillus rhamnosus reduced the incidence of AD while Bifidobacterium animalis did not show any reduction, suggesting the strain dependent preventive effect of probiotics (64). In contrast, two prevention studies did not show any effect on AD incidence. Moreover one of the studies reported an increased sensitization rate for food or aeroallergens in the probiotics group (65,66). There are several possible explanations for these inconclusive results. First, different lactobacillus strains were used and this indicates that not all strains are effective for atopic dermatitis. Second, probiotic effects are dose dependent and there was considerable variation in the daily dosage. The third explanation could be a difference in study design such as numbers and atopic risk of participants and supplementation of the mother during breastfeeding. These studies suggest that careful consideration much be established to decide which probiotics or a mixture of them are employed to treat allergic atopic dermatitis.

Randomized controlled trials to examine the effect of probiotics in the treatment of atopic dermatitis in child also have inconclusive results. Several studies showed a significant reduction of SCORAD (SCORing Atopic dermatitis) in the probiotics treated group (67). Other reports showed that probiotics significantly decreased SCORAD of IgE-associated not general AD (68,69). These reports suggest that only children who at a young age already have IgE sensitization such as elevated total or specific IgE levels get a benefit from probiotics administration. However there are also several studies that did not show any effect of probiotics on the severity or incidence of atopic dermatitis (70,71). There are considerable variations in the probiotic strains and daily dosage and number, age, severity of AD of participants and treatment period, which may result in conflicting results.

Inflammatory bowel disease (IBD) is characterized by a chronic dysregulation of the immune response in the gastrointestinal tract (GI tract). Proinflammatory cytokines such as INF-γ, TNF-α, IL-6 and IL-1β are major pathogenic cytokines but the pathogenesis is still not clear (75). Recently many studies have demonstrated that the GI tracts of patients with IBD are populated with increased levels of adherent and pathogenic bacteria. Fecal numbers of anaerobic bacteria such as Bacteroides species were greater while Lactobacilli and Bifidobacteria were fewer than in healthy control (76,77). Probiotics may be capable of recolonizing the bowel with non-pathogenic strains of bacteria. Probiotics have long been shown to be beneficial in both infectious and non-infectious digestive disorders. Growing evidence indicates that probiotics may be effective in the treatment of specific clinical IBD conditions.

Although it is unclear whether the abnormal composition of the entreric flora contributes to the pathogenesis of IBD, many clinical trials have examined the effect of diverse probiotic strains on the modulation of IBD progression including LGG (78) and E. coli strain Nissle 1917 (79,80). In addition, VSL#3, a mixture of 4 lactobacilli strains (Lactobacillus plantarum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus delbrueckii ssp. bulgaricus), 3 bifidobacteria strains (Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium longum), and 1 strain of Streptococcus (Streptococcus salivarius ssp. thermophilus), has been examined in ulcerative colitis (UC), Crohn's disease (CD) (81,82). The therapeutic effects of several probiotic strains in animal studies and clinical trials were summarized in Table I and Table II, respectively.

Several studies have investigated the influence of probiotic consumption on colitis in animal trials. In particular, IL-10 knockout mouse that develops IBD spontaneously has been largely employed as a IBD model. Interestingly, IL-10 knockout mice spontaneously develop colitis when colonized with a conventional flora but remain disease free when maintained under germ-free conditions (83-87). These studies suggest the role of commensal flora in the development of inflammatory bowel disease. The role of probiotic strains in animal model of IBD was summarized in Table II.

Collectively these studies indicate that although the probiotic administration has potential therapeutic efficacy in IBD, larger controlled trials are necessary before the use of probiotics as a routine medical treatment.