Probiotics, Nuclear Receptor Signaling, and Anti-Inflammatory Pathways

There is increased investigation of the human microbiome as it relates to health and disease. Dysbiosis is implicated in various clinical conditions including inflammatory bowel disease (IBD). Probiotics have been explored as a potential treatment for IBD and other diseases. The mechanism of action for probiotics has yet to be fully elucidated. This paper discusses novel mechanisms of action for probiotics involving anti-inflammatory signaling pathways. We highlight recent progress in probiotics and nuclear receptor signaling, such as peroxisome-proliferator-activated receptor gamma (PPARγ) and vitamin D receptor (VDR). We also discuss future areas of investigation.


Introduction
Probiotics are ingestible microorganisms with health benefits. Increased interest in the intestinal microbiome and its effect on health and disease is evidenced by the concomitant increase in peer-reviewed clinical trials investigating probiotics as therapy since 1999 [1]. Studies of the various signaling pathways involved in the response to bacteria and inflammation have led to a more detailed understanding of mechanisms and actions of probiotics. This paper discusses progress in understanding how probiotics contribute to intestinal mucosal function, particularly in relation to antiinflammatory signaling pathways.

Intestinal Microflora
The intestinal microflora, as a whole, serves important functions in metabolism, intestinal epithelial cell function and health, immunity, and inflammatory signaling [2,3]. Recently, there has been increasing interest in the role of the intestinal microflora and its total genetic composition, together referred to as the microbiome in the development, maintenance, and perpetuation of various clinical conditions, both intestinal and extraintestinal.
Dysbiosis has been implicated in various clinical conditions including atopy, irritable bowel syndrome (IBS), colorectal cancer, alcoholic liver disease in animal and human studies, obesity and other metabolic disorders, and chronic inflammatory diseases such as IBD [4][5][6][7][8][9][10][11]. Decreased diversity of the intestinal microbiota was seen in fecal samples obtained from children who subsequently developed allergic disease [6,7]. Altered microbiota composition in colon cancer patients when compared to patients with normal colonoscopies and in patients with IBS compared to unaffected patients has also been demonstrated [5,9]. Alcohol feeding resulted in enteric bacterial overgrowth in a mouse model [8]. The role of the microbiota in obesity has been extensively studied and carefully reviewed in the literature [12,13]. Microbial composition in IBD patients with ulcerative colitis (UC) or Crohn's disease (CD) as compared to unaffected individuals has been studied and shows decreased diversity [4,[14][15][16][17][18][19]. This altered microflora may have significant implications for the intestinal milieu, with as yet incompletely understood effects. The pathogenesis of IBD 2 Gastroenterology Research and Practice likely involves a combination of factors including intestinal dysbiosis in conjunction with environmental factors in a genetically susceptible host [20].
Based on the concept of a dysregulated or dysfunctional microbiota in disease, various methods to attenuate the effects of an altered microbiome have been attempted.

Probiotics
"Probiotics" were first described in the literature by Lilly and Stillwell in 1965 as growth-promoting factors produced by certain microorganisms [21] although it may have been described as early as 1908 [22]. Recently, probiotics were defined as "live organisms which, when consumed in adequate amounts as part of food, confer a health benefit on the host" (Report of a Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria (October 2001), "Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria", Food and Agriculture Organization of the United Nations, World Health Organization). The mechanisms of action of probiotics include immune modulation, direct effect on commensal and pathogenic bacteria to inhibit infection and restore homeostasis, and modification of pathogenic toxins and host products [23]. The efficacy of probiotics in various clinical conditions both in the pediatric and adult patient population has been extensively studied and carefully reviewed [1,19,[24][25][26][27][28][29][30][31][32].
Rectal infusion of normal stool via enemas to treat pseudomembranous colitis has been described as early as 1958 [70]. Infusion of stool via nasogastric tube to the small intestine or via colonoscopy to the colon for CDAD has also been described and shows high response rates [71][72][73][74]. A recent study showed that fecal bacteriotherapy was effective in relief of clinical symptoms in a patient with recurrent CDAD and that this was accompanied by the repopulation of the diseased intestinal microbiota with beneficial species that were diminished pretreatment [75]. Other methods to supply live, nonpathogenic organisms to the intestinal microbiota in AAD and CDAD include orally administered probiotics. The efficacy of various probiotic formulations in AAD and CDAD has been extensively studied and carefully reviewed [1]. A recent study showed that the probiotics Lactobacillus acidophilus and Lactobacillus casei were well tolerated and effective in reducing the risk of the development of AAD and CDAD [76]. The utility of the probiotic yeast Saccharomyces boulardii for a variety of conditions including traveler's diarrhea, enteral nutrition-associated diarrhea, AAD, and CDAD has been investigated, and according to a recent meta-analysis, strong evidence exists for advocating its use in traveler's diarrhea and AAD [77]. Recent trials using Bifidobacterium bifidum and Saccharomyces boulardii demonstrated improvement in clinical IBS symptoms and quality of life [78,79], and several reviews of the evidence for the utility of probiotics in IBS have been published [80][81][82].
For IBD therapy, treatment with different strains of probiotics has shown varied results. Small trials have shown promise for probiotic use in the induction and maintenance of remission in UC. VSL#3 has been shown to be safe and effective in the treatment of acute mild to moderately active UC [83]. Patients with mild to moderate UC unresponsive to conventional therapy achieved a combined induction remission/response rate of 77% with treatment with VSL#3 [84]. E. coli Nissle 1917 was found to be effective and equivalent to mesalazine in maintaining remission in UC [85]. In another study, Lactobacillus rhamnosus GG (LGG) was equivalent to mesalazine in the maintenance of remission in UC, however, appeared to be more effective in prolonging the relapsefree time [86]. Evidence also exists for the role of probiotics in prophylaxis of pouchitis after surgery in UC patients as well as induction of remission in chronic pouchitis [87,88].
Studies of probiotic use in induction and maintenance of remission and prevention of postoperative recurrence in CD have been less consistent than those for UC. A small study of LGG for the prevention of recurrence after surgery in CD did not show any improvement over placebo [89]; however, Saccharomyces boulardii appears useful in maintaining remission in CD [90,91]. The progress in the use of probiotics for IBD has been carefully reviewed [92,93]; however, there remains a relative lack of well-designed, large, randomized, placebo-controlled trials.
Several barriers exist to advocating broad use of probiotics in clinical practice, not least of which is the considerable heterogeneity in the experimental designs with respect to species and strains of probiotics and the various animal models utilized [94]. Although clinical trials examining the role of probiotics in the treatment and/or prevention of AAD, CDAD, IBD including UC, CD, and pouchitis, necrotizing enterocolitis, infectious gastroenteritis, radiation-induced enteritis, and colitis, IBS and various atopic diseases have been reported [1,24,25,28,29,31,87,[95][96][97]; in many cases, results have been inconsistent, and large, well-designed trials are lacking. An additional complicating factor pertains to issues of quality control. Determining whether a commercially available probiotic actually contains the live organisms it purports to contain and determining if there is rational selection of component probiotic strains in "cocktails" are issues that must be considered [22]. Future research to refine techniques to accurately identify "normal" and "diseased" microbiota and to further elucidate the specific effects and mechanisms of actions of individual probiotic strains will aid in optimizing therapeutic efficacy.

Mechanisms for Probiotics in Anti-Inflammation
There has been and continues to be considerable research in delineating the underlying mechanisms by which probiotics exert their beneficial effects. The mechanisms regulating the function of probiotics are very diverse. It is well accepted that probiotics use distinct cellular and molecular mechanisms, including blocking pathogenic bacterial effects, regulating immune responses, and altering intestinal epithelial homeostasis by promoting cell survival, enhancing barrier function, and stimulating protective responses [32]. Table 1 outlines representative publications on probiotic mechanisms of actions. The probiotic-host interaction is complex and further complicated by the fact that certain probiotic effects appear to be species and strain specific. Different probiotics have been shown to exert both proinflammatory [98] and anti-inflammatory effects on dendritic cells [99]. A recent study demonstrated that the antiinflammatory effect of certain lactobacilli is via NOD2mediated signaling [100]. NOD2/CARD15 is a member of a superfamily of genes involved in intracellular bacterial recognition and has been identified as an important susceptibility gene for CD [101,102]. The authors speculate that the inconsistent clinical results of lactobacilli use in patients with CD may be related to a relative deficiency of NOD2. Probiotic effect on the innate immune responsive pathways including toll-like receptor (TLR), nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK) has been extensively investigated ( Table 1). Activation of specific TLRs also appears to be species specific [47,48]. The action of E. coli Nissle 1917 on Caco-2 cells was found to be mediated by flagellin possibly via a TLR pathway [103]. The probiotic-induced effect on the NF-κB signaling pathway is well represented in the literature and is generally characterized by inhibition (Table 1).
Defective epithelial barrier function has been implicated in IBD and can predict relapse during clinical remission [104][105][106][107][108][109]. One way by which probiotics have been shown exert their action is by stabilizing tight junctions (TJs) and enhancing barrier function of intestinal epithelial cells (Table 1).
Abnormal STAT/suppressor of cytokine signaling (SOCS) signaling has been demonstrated in CD patients [110], and probiotics are also shown to modulate the JAK-STAT signaling in human placental trophoblast cells [111]. Increasing evidence further demonstrates that metabolism, xenobiotics, and nuclear receptor signaling are involved in the action of probiotics [67,68].
Induction of heat shock proteins (HSPs) and endogenous antimicrobial peptides (defensins) via activation of NF-κB, MAPK, and JNK has also been linked to probiotic action [35,41,43]. Since defensins are implicated in the pathogenesis of IBD, increased expression by probiotics provides a possible mechanism for clinical efficacy seen in certain IBD patients and deserves further study.

Defensins and Nuclear Receptor Signaling
Defensins are a class of endogenous antimicrobial peptides involved in innate immunity which is highly evolutionarily conserved and represents a primary line of defense against various microbial pathogens [112][113][114]. Antimicrobial peptides are widely distributed throughout the animal and plant kingdom, and despite their evolutionary heritage, remain effective antimicrobial agents [114]. This is due, in large part, to their mechanism of action involving membrane disruption and pore formation, which is not easily exploited by pathogens to confer resistance [112][113][114][115]. Important antimicrobial peptides in humans include defensins, cathelicidins, lysozymes, and other antimicrobial antiproteases [116]. There are three known defensin subfamilies; α and β defensins are expressed mainly in immune cells and epithelial cells while the θ defensin is found mainly in immune cells of the Rhesus macaque [117,118]. In the gastrointestinal tract, β defensin expression is seen in multiple sites, whereas α defensin expression is largely in the small intestine [119]. In the uninflamed colon, human β defensin 1 is the predominant defensin and human β defensin 2 and 3 are induced with inflammation or infection [120]. In mice lacking functional cryptidins (murine α defensins), increased survival and virulence of orally administered bacteria were seen and intestinal peptide preparations had decreased antimicrobial activity [121].
The possible role of a deficiency in defensins in the pathogenesis of IBD has been proposed [116,122]. The Paneth cells of the small intestine are the major source of endogenous antimicrobials, including α defensins [102]. In addition, The Paneth cells have been shown to express NOD2 [123]. In patients with ileal CD, human α defensin 5 and 6 production is reduced, and this effect is magnified in those patients with a concomitant NOD2 mutation [124]. For β defensins, CD patients with colonic disease exhibit normal levels of β defensin 2 and 3 whereas UC patients have increased levels, suggesting a role of failure of β defensin induction in the pathogenesis of CD [125]. Constitutive human β defensin 1 expression is reduced in CD patients with colonic involvement independent of inflammation, and recently, the maintenance of constitutive β defensin expression was shown to be activated by the nuclear receptor peroxisome-proliferator-activated receptor gamma (PPARγ) [122].
Further contributing to the effect of a defensin deficiency in the pathogenesis of IBD may be the diminished diversity of the intestinal microbiota seen in IBD patients. The interaction of commensal bacteria with antimicrobial peptide synthesis is not well understood; however, it has been suggested that commensal bacteria provide chronic stimulation of epithelial cells to produce antimicrobial peptides at levels sufficient to kill microbial pathogens [114,126].
Interestingly, nuclear receptors are known to regulate the expressions of defensins [122,127]. Nuclear receptors represent a class of intracellular transcription factors activated by ligands which can directly interact with DNA; as a result, nuclear receptors play significant roles in the regulation of metabolic, reproductive, developmental, and immune processes [128][129][130][131]. Nuclear receptors regulate transcriptional activity by several distinct mechanisms, including "liganddependent transactivation, ligand-independent repression, and ligand-dependent transrepression" although the range of transcriptional activities of each nuclear receptor varies and even the transcriptional effects of a single nuclear receptor may be cell specific [132]. A detailed discussion Table 1: Summary for molecular mechanisms and probiotics.

Involved pathways Probiotics
In vitro system NF-κB LGG reduced TNF-α-induced NF-κB translocation, lessened decrease in IκB, reduced TNFα-induced interleukin (IL)-8 production [34] NF-κB   (iii) Cytokine secretion was almost undetectable and not modulated by ECN in TLR-4-knockout mice (iv) Increased TLR2 and TLR4 protein expression and NF-κB activity via TLR2 and TLR4 was seen with ECN and human T-cell coculture [48] 6 Gastroenterology Research and Practice      (v) The effects of ST/La or BT on TER and permeability were potentiated by a Janus kinase (JAK) inhibitor but not by p38, ERK1, 2, or PI3K inhibition (vi) Reduced activation of suppressor of cytokine signaling (SOCS)3 and STAT1,3 was seen only in probiotic-treated epithelial cells exposed to cytokines [63] JAK/STAT NF-κB   (iii) Lipid-and PPAR-signaling-associated genes were also upregulated (iv) Altered microbial diversity was noted in probiotic-treated mice (v) Inflammation in IL10-KO mice showed differential regulation of various signaling pathways( inflammatory, nuclear receptor, lipid, and xenobiotic) [67] Metabolism/ Glucose Uptake LGG prevented TNF-α-induced activation of intestinal NF-κB [69] of nuclear receptors and their mechanisms of action is beyond the scope of this article; however, further discussion of two nuclear receptors (peroxisome-proliferator-activated receptor gamma (PPARγ) and vitamin D receptor (VDR)) with putative roles in inflammation is warranted. PPARγ is a member of a class of nuclear receptors that form obligate heterodimers with the retinoid X receptor (RXR) [129]. The PPAR family has been shown to affect various cellular functions including "adipocyte differentiation, fatty-acid oxidation, and glucose metabolism" [129]. PPARγ is highly expressed in the large intestine [133], and its activation has been shown to be protective in animal models of colitis [134,135]. Decreased PPARγ expression in UC patients has been shown [136], and the anti-inflammatory compound 5-aminosalicylic acid (5-ASA) commonly utilized in IBD therapy was shown to be a PPARγ agonist, thereby establishing a possible mechanism by which it exerts its anti-inflammatory effects [137]. PPARγ also plays a role in the maintenance of "constitutive epithelial expression of a subset of β defensins in the colon" [122].

Vitamin D and Vitamin D Receptor (VDR)
Vitamin D receptor (VDR) is a nuclear receptor that mediates most known functions of 1,25-dihydroxyvitamin D (1,25(OH) 2 D 3 ), the active form of vitamin D [138]. VDR heterodimerizes with RXR once VDR is activated by 1,25(OH) 2 D 3 . VDR binds to the vitamin D response element in the target gene promoter to regulate gene transcription [139]. VDR downstream target genes include antimicrobial peptides such as cathelicidin and β defensin.
VDR is critical in regulating intestinal homeostasis by preventing pathogenic bacterial invasion, inhibiting inflammation, and maintaining cell integrity [140][141][142][143][144][145]. Vitamin D directly modulates the T-cell receptor (TCR) [146], and vitamin D has also been shown to downregulate the expression of proinflammatory cytokines and have regulatory effects on autophagy and various immune cells including T cells, B cells, macrophages, dendritic cells, and epithelial cells [147,148]. It has been reported that 1,25(OH) 2 D 3 suppresses the development of IBD in animal models [149]. Deficiency of 1,25(OH) 2 D 3 has been reported in patients with IBD [150,151], and, recently, using a novel vitamin D bioavailability test, vitamin D deficiency or insufficiency was seen in more than 70% of patients with quiescent CD [152]. Given the diverse immune functions of vitamin D, deficient levels may have important implications for the development and maintenance of intestinal homeostasis. A possible role of vitamin D status and VDR signaling in modulating the effects of intestinal microflora in other conditions such as asthma and obesity has been suggested [100]. While present literature has primarily focused on elucidating the immunoregulatory effects of vitamin D, there is a paucity of data on the status and function of VDR [147]. In addition, probiotic-induced modulation of anti-inflammatory VDR signaling in colitis remains virtually unexplored.
Recent studies indicate that VDR−/− mice have increased bacterial loading in the intestine [145,153]. Our microarray data found that VDR signaling responds to pathogenic Salmonella in intestinal colitis in vivo [154]. Data from a recent study demonstrate that bacterial stimulation, both commensal and pathogenic, regulates VDR expression and location and that VDR negatively regulates bacterialinduced intestinal NF-κB activation [153]. In general, probiotic-induced nuclear receptor signaling is not well characterized. The probiotic VSL3# was associated with nuclear receptor signaling in the IL10−/− colitis model [67]. Nuclear receptors have been shown to negatively regulate bacterialstimulated NF-κB activity in intestinal epithelium [153,155]. Our recent data show probiotic treatment is able to enhance VDR expression and activity in the host. An increase in VDR expression and a concomitant increase in cathelicidin mRNA in cultured intestinal epithelial cells when treated with Lactobacillus plantarum were seen [156]. We used a probiotic monoassociated pig model to assess the probiotic effect on VDR expression in vivo and found intestinal VDR increased significantly after probiotic colonization compared to the ex-germ-free pig. Furthermore, our unpublished data indicate that probiotics did not inhibit inflammation in mice lacking VDR.
The presence of VDR in various tissues along with its ability to exert diverse actions in differentiation, growth, and anti-inflammation sets the stage for exploitation of VDR ligands for the treatment of various inflammatory conditions [157,158]. Although the potential importance of VDR as a therapeutic target has been appreciated [159], no approach to date has safely and effectively altered VDR's activity. Hence, understanding VDR's contribution to probioticinduced anti-inflammation may provide significant insight in the pathogenesis of inflammatory conditions such as IBD, and thereby, guide the development of novel treatments. Further investigation of the complex interplay of nuclear receptors, defensins, probiotics, and inflammatory pathways may provide significant insight into the mechanisms of action of probiotics in anti-inflammation.

Current Problems and Future Directions
The individual diversity of the intestinal microflora underscores the difficulty of identifying the entire human microbiota and poses barriers to this field of research. In addition, it is apparent that the actions of probiotics are species and strain specific [19]. It is also apparent that even a single strain of probiotic may exert its actions via multiple, concomitant pathways. Current investigation into the mechanism of action of specific probiotics has focused on probioticinduced changes in the innate immune functions involving TLRs and its downstream systems including NF-κB, JAK-STAT, MAPK, and SAPK/JNK pathways. Future research on novel mechanisms of action for probiotics involving nuclear receptor signaling, including PPARγ and VDR, is needed. With evolving knowledge, effective probiotic therapy will be possible in the future.