Mutualism between a commensal microbiota and its mammalian host primarily reflects the evolution of a relationship that offers metabolic advantages to the host. Although these commensal organisms reside mainly in the gut, the physiological effects of mutualism connect the gut to distant tissues, such as muscle, liver, and brain.1 In the gut host tissue, external factors like diet and the microbiota operate as an intricate triumvirate, defining the immediate and distant milieus and thereby shaping various physiological and disease processes. In this triumvirate, each partner's existence is in harmony with that of the other 2 (Fig. 1). Manipulations of any partner in this system would be expected to have a profound impact on the others by disrupting that harmony. This expectation, in theory, forms the basis for work aimed at altering host physiology through manipulation of the commensal microflora.
The notion that microbial species affect host physiology is supported by reports that different microbial species inhabit the host at different stages of its life. For example, the influence of early life processes on the human microbiota include the type of delivery chosen for the mother: Lactobacillus, Prevotella, and Atopobium are prevalent in vaginally delivered neonates, whereas staphylococci and other members of the maternal skin community are prominent in infants delivered by cesarean section.1 A study of healthy neonates in Greece found a lower prevalence of Lactobacilli and Bifidobacteria on days 4 and 30 in cesarean-delivered than in vaginally delivered infants.2 Moreover, altered proportions among bacterial species have been associated with disease states. Examples include an association of Lactobacillus, Bacteroides, and Firmicutes with inflammatory bowel disease (IBD) and Bacteroidetes with weight loss.3–5
As a major element of the third member of the triumvirate, diet has an enormous effect on the composition of the microbiota, influencing host physiology both directly and indirectly. Thus, manipulation of the microbiota through dietary changes, for example, has significant therapeutic potential in patients suffering from, for example, IBD.6,7
ROLE OF THE COMMENSAL MICROBIOTA IN ANTI-INFLAMMATORY/IMMUNOREGULATORY ACTIVITY
Inflammatory processes are critical mechanisms that enhance immunity against invading pathogens and damaged tissue. These processes influence both the innate and the adaptive arms of the immune system. In several tissues, however, prolonged inflammation itself leads to tissue damage and engenders a pathological outcome. To avoid this deleterious outcome and restore homeostasis, anti-inflammatory or immunoregulatory processes are recruited. The factors governing such immunoregulatory processes have yet to be fully elucidated. However, evidence for the generation or expansion of immunoregulatory responses is gradually accumulating.
Microbiota-sensitive Immunoregulatory T Cells
A role for regulatory T cells (Tregs) reactive to the intestinal microbiota in cross-protection against certain pathogenic antigens has been suggested. In a study of such cross-protection, mice were fed trinitrophenyl (TNP)–haptenated colonic proteins obtained after homogenization of colonic specimens and subsequent TNP haptenization with 2,4,6-trinitrobenzene sulfonic acid (TNBS).8 TNBS-induced colonic inflammation is an established animal model of IBD, wherein pathogenicity is attributed both to innate myeloid cells and to Th1 and Th17 cells and results from precipitation of pro-inflammatory reactions by haptenated self-antigens and non–self-antigens. The investigators observed protection against TNBS colitis in mice fed with TNP-haptenated colonic proteins. In addition, they observed expansion of the Treg population producing transforming growth factor β (TGF-β) and interleukin 10 (IL-10) in the lamina propria of mice fed with TNP-haptenated colonic proteins; subsequent transfer of these lamina propria Tregs protected recipient mice. When the researchers went on to analyze the Tregs, they found that TGF-β–producing activity played a primary role in protection and that IL-10 activity helped to stimulate this primary protective function. Such cross-reactive Treg function may reflect oral tolerance induction, illustrating the potentially tolerogenic nature of the oral route of administration that has been elaborated elsewhere.9 Boirivant et al8 demonstrated that the bacterial probiotic VSL#3 (which contains Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus bulgaricus, and Streptococcus thermophilus) significantly reduced the second induction of TNBS colitis by generating similar TGF-β–bearing, IL-10–dependent Tregs in the colonic lamina propria. Further elegant studies confirmed this capacity of bacterial antigens to generate Tregs. FOXP3+CD4+ Tregs were shown to be abundant in the colonic mucosa10; Clostridium species (particularly clusters IV and XIVa) were found to cause the accumulation of these cells. Consecutive conventionally reared mice developed resistance to both colitis and systemic IgE responses after oral administration of clostridia during early life.
A prominent example of an immunomodulatory organism is the human gut commensal Bacteroides fragilis, an obligate anaerobe uncommon in the murine gut flora. Monocolonization of gnotobiotic mice with B. fragilis resulted in immune system maturation from a TH2 phenotype seen in germ-free mice, into a balanced TH1/TH2 phenotype.11 In addition, B. fragilis treatment of mice led to profound resistance to inflammatory disease in both the gut and distant tissues.12,13 In animal models of colitis and multiple sclerosis, the ability of CD4+ T cells to generate the potent anti-inflammatory cytokine IL-10 was found to underlie the protective function of the organism. Bacteroides fragilis expresses 8 polysaccharides (designated A through H) in its capsule. In expressing so many different polysaccharides, B. fragilis is unique among the microflora species in the mammalian gut. Genes for the B. fragilis polysaccharides are under the control of different polysaccharide biosynthesis locus promoters whose particular orientation can give rise to expressing one or occasionally more of the polysaccharide capsules.14 Exploitation of this feature has led to the development of an important experimental tool: a B. fragilis mutant strain expressing only polysaccharide A (PSA). Interestingly, both the immunostimulatory and the immunoregulatory activities of B. fragilis are a function of PSA. Moreover, in a classic example of host–commensal interaction orchestrated by immunological processes, PSA interacts with the immune system to allow B. fragilis to colonize niche areas in the mammalian colon.15 (PSA's mechanism of action is discussed later in this review.) However, the immunomodulatory activity of B. fragilis may not be entirely due to PSA alone. Bacteroides fragilis also expresses sphingolipids—immunomodulatory molecules primarily described on eukaryotic cells.16 Whether these sphingolipids confer immunomodulatory properties in settings of inflammatory disease remains to be investigated.
Applicability of Treg Data from Murine Models to Patients with IBD
Tregs directed against specific microbial antigens, host antigens, or even stochastic dietary antigens may have therapeutic applications in IBD.17 The principal role these cells play in immunosuppression also raises the possibility that dysregulation in the Treg compartment is a causal element in the undefined etiology of IBD. FOXP3 has been found to be absent or nonfunctional in some patients with severe intestinal inflammation.18,19 On a more mechanistic level, it has been demonstrated in mice that a deficiency in Tregs marked with FOXP3, in conjunction with deficiencies in functional immunoregulatory molecules known to be immunosuppressive (e.g., IL-10, IL-35, and TGF-β), causes severe colonic inflammation.20–24 Nevertheless, the correlation of Treg deficiency with disease in patients has not been a foregone conclusion; doubts persist about whether Treg functions observed in vitro accurately reflect Treg functions in patients. However, it has been demonstrated that Tregs isolated from the blood and the intestinal mucosa of patients with IBD are suppressive in vitro.25,26 In addition, increased Treg numbers in both inflamed and uninflamed gut tissue sections from patients with IBD indicate a probable redundancy in known Treg functions26–28; it can also be argued that Tregs are in the process of controlling inflammation in the still-inflamed portions of gut biopsy samples and that inflamed and uninflamed tissues are separated temporally in terms of restitution or resolution. Whatever the case may be, clinical data lead us to think beyond the information obtained in studies of laboratory animals, including murine models of inflammatory disease. Clearly, a Treg-driven approach to potential therapies for human disease must be pursued with caution.
Apart from the Tregs that are selected for antigenic specificity in the thymus (designated nTregs), Tregs can also be induced at the periphery—i.e., at a site outside the thymus—as a consequence of certain selection pressures. A classic site for the induction of Tregs is the gut itself. Induced Tregs (iTregs) have been shown to prevent T cell–driven colitis in mouse models.24 iTregs can be generated in vitro by stimulation (e.g., by TGF-β or rapamycin) or by the action of tolerogenic dendritic cells (DCs).29,30 In addition to classical immunostimulation, tolerogenicity has been described as a fundamental—albeit not exclusive—property of DCs. In the presence of antigen and other factors, immunoregulatory properties in T cells can be induced by almost any DC. However, some subsets of DCs reportedly have greater tolerogenic potential than others and function as “instructors” for Tregs in steady-state or inflammatory situations. Worthy of special mention are the classic DCs in the gut that express CD103 and function primarily through the action of retinoic acid–converting enzymes. Contrasting evidence regarding tolerogenic function has been reported in the setting of inflammation. On the one hand, retinoic acid, CD103 DCs, and DCs displaying the receptor for CD103 (E-cadherin) have been found to promote inflammation31–33; on the other hand, a tolerogenic role for these DCs in colonic inflammation has been observed.34,35 Plasmacytoid dendritic cells (PDCs), another subset, have been credited with protective roles in murine models of disorders, such as graft-versus-host disease, airway inflammation, and multiple sclerosis.36–39 Although PDCs are known primarily for liberating the pro-inflammatory cytokine type 1 interferon in response to viral or bacterial nucleic acids, their role in oral and central tolerance has also been demonstrated.40,41
Antigen-specific Tregs, including FOXP3− IL-10–producing Tr1 cells, have also been considered for the treatment of colitis. Ovalbumin-specific Tr1 cells were effective in protection against murine colitis and—importantly—in the treatment of refractory Crohn's disease.17 The ovalbumin specificity of these cells was exploited to induce an in vivo anti-inflammatory mechanism that probably affected all types of pro-inflammatory T cells. The expansion of intestinal iTregs in vivo, which has already been successfully undertaken, may provide a better therapeutic approach to in vitro generation and in vivo transfer of iTregs.42,43
Another issue in the clinical application of Tregs has to do with a lack of success in the administration of IL-10 itself.44,45 The very short half-life of administered IL-10 may explain its lack of effectiveness. Delivery of IL-10 by improved means might circumvent this problem. In this regard, adenovirus-transfected IL-10 gene delivery has been shown to protect mice from colitis.46,47 Genetically modified bacteria offer another alternative: delivery of IL-10 by this route has been tested in humans, with Lactococcus lactis as the host bacterial species engineered to express this cytokine.48
In short, use of inherent immunoregulatory properties in selected commensal bacteria or of genetically engineered immunoregulatory properties in these organisms is one approach to the treatment of IBD in the future.
FACTORS IN INFLAMMATION
Increased levels of pro-inflammatory cytokines—namely, interferon gamma (IFN-γ), IL-12, IL-17, and IL-18 in patients with Crohn's disease and IL-13 in patients with ulcerative colitis—suggest the involvement of helper T-cell subsets Th1 and Th17 in the former disease and of the Th2 subset in the latter.49–55 In addition, a significant role for the intestinal microflora and genetic susceptibility in the induction of IBD has been demonstrated.56 Important evidence for the role of the commensal microbiota in this disease has come from the isolation of T-cell clones specific for species of organisms such as the Enterobacteriaceae, Bacteroides, and Bifidobacterium.57 Murine models of colitis have yielded evidence for the pathogenicity of T cells specific for bacterial flora. For example, spontaneous colonic inflammation resulted when CD4+ T cells specific for clostridial flagellin were transferred to immunodeficient C3H/HeJBir mice.58,59 Moreover, in recent years, commensal segmented filamentous bacteria (SFB) have been regarded as a major factor in the pro-inflammatory axis in murine systems and have been linked to inflammation-induced pathology. Colonization of germ-free mice with SFB induces lamina propria CD4+ T cells that produce the pro-inflammatory cytokines IL-17 and IL-22; the result is enhanced resistance to the intestinal pathogen Citrobacter rodentium.60 In addition to enhancing mucosal immunity, SFB are associated with inflammatory diseases, such as experimental autoimmune encephalomyelitis.61
A skew in the commensal microbiota—in particular, a reduction in the gram-positive flora and an increase in the gram-negative flora relative to values in healthy individuals—has been observed in patients with IBD.62 Thus, metagenomic analysis of bacterial candidate genes, even those from animal models of gut inflammation, is a potential means of linking specific molecules to disease pathogenesis. Immune dysfunction leading to inflammatory disorders is observed in elderly individuals; investigators are assessing the link between changes in the microbial flora and age-related chronic inflammatory diseases.63
Bile acids are important metabolic components of the mammalian gastrointestinal system. Searching for factors in gut inflammation, Duboc et al64 recently investigated perturbation of the microflora and the impact of this perturbation on bile acid metabolism. The researchers observed that a decreased ratio of Faecalibacterium prausnitzii to Escherichia coli was a marker for IBD-associated dysbiosis. Importantly, certain microbiota-mediated aspects of bile acid metabolism, including deconjugation, transformation, and desulfation, were dysfunctional in patients with active IBD, who were compared with healthy volunteers; a consequence of this dysfunction was the accumulation of fecal 3-OH-sulfated bile acids. This change in the luminal bile acid pool may have important implications for gut inflammation: sulfated secondary bile acid species were devoid of in vitro anti-inflammatory properties. In a classic case, the influence of dietary fat on a particular pathobiont was recently demonstrated; fat-induced expansion of the growth of the sulfite-reducing species Bilophila wadsworthia exacerbated colitis in IL-10–knockout (IL-10 KO) mice.65 A diet rich in milk-derived saturated fat induced taurine conjugation of bile acids, increasing the availability of sulfur for use by the pathobiont. Another molecule that is important to metabolism and thought to influence homeostasis is H2. Given the beneficial role of H2 (especially in preventing oxygen-induced damage), it has been proposed that a balance between H2-producing and H2-using bacteria might influence immune homeostasis in the colon.66
Some host immune molecules have important effects on microbial colonization in the gut.67 Recently, a member of the innate immune-sensing repertoire—namely, Toll-like receptor 5 (TLR5), a receptor for bacterial flagellin—was shown to link the microbiota to the development of colitis in mice.68 The difference in microbiota between colitic TLR5-deficient (TLR5 KO) mice and wild-type mice was long-lasting, whereas in noncolitic TLR5 KO mice, the difference was transient. The colitic microbiota included high levels of Proteobacteria—especially enterobacterial species, including E. coli. In addition, a Crohn's disease–associated strain of E. coli caused long-term chronic inflammation after administration to TLR5 KO mice; this inflammation persisted even after the bacteria had been eliminated from the system. Another host molecule, (peptidyl-dipeptidase A) 2 or ACE2, has a gut microbiome–regulating function implicated in intestinal inflammation. Microbial transplantation from ACE2-deficient mice to wild-type germ-free recipients increased the propensity of the recipients to develop severe colitis.69 As observed in IL-10 KO mice, the role of IL-10 in suppressing inflammation is evident in spontaneous colitis; these mice provide an elegant platform on which to investigate the role of the microbiota and diet in upregulating or downregulating colitis in an IL-10–independent manner.
STUDIES OF BACTERIA FROM PATIENTS WITH IBD
Information from patients with pediatric IBD is useful for understanding underlying disease mechanisms in humans. In a substantial proportion (20%–25%) of patients with IBD, disease has its onset during childhood.70 Microbial dysbiosis is a major factor in IBD in children, as it is in adults. Genetic predisposition is another critical factor in pediatric IBD. Interestingly, several genes that are related to microbial recognition and the innate immune pathway [e.g., nucleotide oligomerization domain 2 (Nod2)] and genes that mediate autophagy (e.g., ATG16L1, IRGM) have been implicated in IBD.71 Adherent-invasive E. coli, whose enhanced adhesive and invasive properties are attributable to the mannose-binding FimH protein, is associated with pediatric IBD.72 Further characterization of mucosa-associated E. coli in children revealed another 11 strains with a high degree of fimH sequence similarity. Interestingly, neuraminidase treatment—whose effects mimic mucosal inflammation—enhanced adhesion of all 11 strains by 3.5-fold; this enhancement indicated that these strains might be able to persist in an inflamed environment and participate in cumulative inflammation-induced pathology in the host.
In a study correlating bacteria with early-stage Crohn's disease in Norwegian patients, metagenomic analysis revealed 106 different bacterial operational taxonomic units in the cloned libraries generated from mucosal biopsies.73 The operational taxonomic units belonged mainly to the Bacteroidetes (42% in patients versus 71% in controls) and the Firmicutes (42% in patients versus 28% in controls). The rest of the operational taxonomic units belonged to the Proteobacteria (15% in patients versus 0 in controls) and unassigned phyla.
The variety of the gut microbiota increases with age. Claesson et al74 observed that diet-based grouping of individuals correlated with residence location and microbiota composition, which further correlated with markers of inflammation. In addition, the microbiota was less diverse in individuals who were in long-stay-care facilities than in community dwellers, and this loss of microbial species was correlated with increased physical weakness.
Pre-existing IBD leads to higher morbidity and mortality following Clostridium difficile infection (CDI).75 Furthermore, when patients with ulcerative colitis with or without CDI are compared, those with CDI are found to be more vulnerable to health problems, as evidenced by more ulcerative colitis–related emergency room visits in the year after initial infection (37.8% versus 4%, P < 0.001), more colectomies (35.6% versus 9.9%, P < 0.001), and increased medical therapy and disease severity.76 Some common infections like that with Helicobacter pylori are less frequently associated with IBD.77 In patients who underwent same-day bidirectional gastrointestinal endoscopy and biopsy, the presence of H. pylori was inversely associated with IBD, with adjusted odds ratios of ∼0.5 for Crohn's disease, ulcerative colitis, and intermediate colitis, i.e., an IBD case diagnosis of which cannot decide whether the case is Crohn's disease or ulcerative colitis. In contrast, H. pylori–negative gastritis was positively associated with IBD [adjusted odds ratios (95% confidence intervals): 11.06 (7.98–15.02) for Crohn's disease, 2.25 (1.31–3.60) for ulcerative colitis, and 6.91 (3.50–12.30) for intermediate colitis].
IBD outcome may be further complicated by intestinal superinfection. Antonelli et al78 recently reported flare-ups related to intestinal infections in 13.3% of patients; of the total of 143 flare-ups, 12% were associated with infective agents, such as Campylobacter jejuni, C. difficile, and cytomegalovirus. IBD pathogenesis may be caused not only by bacteria but also by viruses, among which enteropathogenic viruses are the prime suspects. However, in a clinical study of patients with IBD visiting the outpatient clinic of the Maastricht University Medical Center, no correlation of either Crohn's disease or ulcerative colitis with enteropathogenic viruses was found.79 This result ruled out an obvious implication of these viruses in IBD.
Disease at a Different Tissue Site
The effect of IBD on the microbiota at a site outside the gut was reported recently. Patients with or without IBD who had chronic periodontitis were analyzed for microbiota at sites of gingivitis and periodontitis.80 At both sites, patients with Crohn's disease had significantly higher numbers of bacteria (a population including Bacteroides ureolyticus, Campylobacter gracilis, Parvimonas micra, Prevotella melaninogenica, Peptostreptococcus anaerobius, Staphylococcus aureus, Streptococcus anginosus, Streptococcus intermedius, Streptococcus mitis, Streptococcus mutans, and Treponema denticola) than did patients with ulcerative colitis. Prevotella melaninogenica, S. aureus, S. anginosus, and S. mutans were more numerous at both sites in patients with Crohn's disease than in controls, whereas counts of S. aureus and P. anaerobius were higher in patients with ulcerative colitis than in controls but only at sites of gingivitis.
Hepatic encephalopathy is a disease originating in the liver in which an altered level of consciousness leads to coma. A difference in microbiota in patients with hepatic encephalopathy was recently documented in a study comparing sigmoid mucosal biopsy samples and stool samples by multitag pyrosequencing.81 The load of pathogenic bacteria was significantly greater and the load of beneficial autochthonous bacteria (e.g., Blautia, Roseburia, Faecalibacterium, and Dorea) was significantly reduced in patients with cirrhosis and hepatic encephalopathy from the corresponding values in control volunteers. In addition, patients with hepatic encephalopathy had worse liver disease scores and higher levels of the inflammatory cytokine IL-6 and endotoxin than did patients without hepatic encephalopathy. Interestingly, although the stool microbiota did not differ between patients with hepatic encephalopathy and those without, hepatic encephalopathy patients had more abundant numbers of Enterococcus, Veillonella, Megasphaera, and Burkholderia and lower counts of Roseburia species.
THERAPEUTIC AGENTS DERIVED FROM THE COMMENSAL MICROBIOTA
The organisms of the commensal microflora possess a strong and varied repertoire of functions important in manipulating human physiology to better prepare for pathological assault or to better manage patients undergoing such trauma. However, several difficulties are encountered in designing interventions based on this microflora. First, variations in the commensal microflora with geography, culture, diet, and even parameters like time or the age and sex of the individual patient make the choice of components for manipulation extremely complicated. Second, selection of a particular function to target is difficult, given that the ultimate functioning of a complex flora is not merely the sum of the functions of its individual members but more likely reflects a “vectorial addition,” wherein contribution of “sense” is paramount. Third, the deliverability of a functional unit consisting of living entities is complicated by possible alterations in properties and activities during storage or at some other point before use. Finally, investigations of the commensal microflora, its components, and their functionalities at the molecular level are in their nascent stage, and years of research must be done before microflora-based molecular therapeutics can be delivered at clinics.
Approaches that have been tried in experimental mice and/or in preliminary human trials include fecal transplantation (i.e., transfer of whole feces—a complex flora—or of fecal bacteria to a recipient from a donor), use of probiotics (i.e., transfer of one or several selected bacterial species—usually cultured organisms—to a recipient), and transfer of relatively pure microbial molecules from selected members of the commensal microflora to a recipient (Fig. 2). These approaches add natural or biological options to the repertoire of existing approaches of application of immunosuppressive drugs, which are often plagued by lack of efficacy and deleterious side effects.
Restoring the microflora to a state of eubiosis from the dysbiosis that leads to IBD is the primary aim of fecal transplantation. In rudimentary form, this approach has been used for the past 1700 years.82 Although fecal transplantation has been used less frequently during the past 50 years, its use to treat CDI epidemics and also some idiopathic conditions has increased in the past few years.83 Case reports have described successful treatment of refractory CDI and ulcerative colitis with fecal transplantation.84
Our knowledge of the effects of a single or a group of bacterial species on intestinal homeostasis is expanding. Although it is not clear what precipitates bacteriologically complex diseases like IBD, diseases like CDI-induced pseudomembranous colitis and diarrhea provide information supporting the effects of dysbiosis on pathogenesis. The fact that CDI can be effectively treated with fecal transplantation strengthens the concept that certain bacterial populations favor intestinal homeostasis. Studying a cohort of 74 patients with recurring CDI at a single institution in northern Minnesota, Rubin et al85 reported a 79% cure rate after nasogastrically delivered fecal transplantation. Interestingly, even in the majority of the cases in which relapses occurred, resolution followed a single dose of vancomycin. Cure rates of >90% have also been reported.86 Use of deep-sequencing techniques after fecal transplantation in patients with CDI has revealed changes in the microbiota, especially favoring Bacteroidetes and Firmicutes, which may serve as probiotics in the future.87 The recent eradication of CDI in an ulcerative colitis patient receiving immunosuppressive treatment opens new vistas for the use of this procedure.88 As successful trials are reported, fecal transplantation during colonoscopy is also gaining ground as an alternative.89 Nevertheless, doubts exist about the use of fecal transplantation as a viable therapeutic approach.90 Certainly, poor screening of donors can be an impediment in the management of patients undergoing fecal transplantation, resulting in the transmission of latent infections.
Application of fecal transplantation in IBD has been reported only rarely (i.e., in 3 studies encompassing 8 cases of ulcerative colitis and 1 case of Crohn's disease), and the use of animal models to study fecal transplantation in IBD have been rarer still.84 However, use of fecal transplants from genetically depleted mice (e.g., the TRUC strain, which is deficient in T-bet and Rag, or ASC protein–deficient animals) to their wild-type counterparts or between wild-type mice from 2 different sources (Taconic Farms, New York, NY and Jackson Laboratory, Bar Harbor, Maine, respectively) has identified a pro-inflammatory role for several microorganisms, including Proteus mirabilis, Klebsiella pneumoniae, Prevotella, bacteria of the TM7 lineage, and SFB.60,91–94
Another approach to manipulation of the microbiota to alter physiological processes is the delivery of one or a combination of bacteria as probiotic therapy.95 Bacterial spores have been used in murine models to determine their anti-inflammatory properties.96 Spores from Bacillus subtilis strain PB6 (ATCC PTA 6737) in powdered form were used to treat animals undergoing TNBS induction of colitis. In addition, this strain enhanced the production of IL-10 in vitro; decreased the production of the pro-inflammatory cytokines IL-12, tumor necrosis factor–alpha, and IFN-γ on human peripheral blood mononuclear cells in vitro; and prevented colitis in mice.
Gut permeability is a crucial factor in initiation of the inflammatory process that leads to IBD. In assessing this factor, timing of measurement is important. Leber et al97 reported increased gut permeability in patients with the metabolic syndrome but found very little change in pro-inflammatory factors. In addition, probiotic treatment with Lactobacillus casei strain Shirota failed to alleviate the metabolic syndrome in these patients.97 Another species, Lactobacillus salivarius Ls-33, was recently tested—without success—as a probiotic in the context of the metabolic syndrome.98 Delivery of Lactobacillus reuteri (ATCC 55730) by enema had therapeutic consequences when administered in conjunction with oral mesalazine to 6-year-old to 18-year-old children with ulcerative colitis.99 Compared with the placebo-treated group, the probiotic-treated group had reduced Mayo scores (including clinical and endoscopic features) and histological scores. At the molecular level, post-trial concentrations of mucosal pro-inflammatory cytokines (IL-1β, tumor necrosis factor–alpha, and IL-8) decreased significantly, whereas concentrations of the anti-inflammatory cytokine IL-10 increased significantly.
Probiotic-induced modulation of intestinal cell death has been suggested as a mechanism of action for this group of therapeutics in IBD.100 Lactobacillus rhamnosus GG inhibited tumor necrosis factor–alpha–mediated apoptosis of intestinal epithelial cells.101 Soluble factors p40 and p75 released by these bacteria were responsible for cytokine-mediated apoptosis induction.102 Whatever the mechanism, the results of clinical trials with L. rhamnosus GG have been mixed at best.103,104 In contrast, the pro-apoptotic effects of E. coli strain Nissle 1917 on γ∂ T cells may hinder the perpetuation of inflammation mediated by this T-cell subset, establishing homeostasis.105 This effect may be clinically relevant: evidence for increased numbers of γ∂ T cells was found in the blood of patients with IBD.106 A mechanism by which the probiotic VSL#3 induces protection in murine colitis was recently demonstrated.107 Myeloid cell–associated peroxisome proliferator–mediated receptor gamma was targeted by probiotic-generated conjugated linoleic acid engendering the anti-inflammatory effect. In consecutive peroxisome proliferator–mediated receptor gamma–deficient animals, no such protective effect of the probiotic was observed.
In a rat model of neonatal necrotizing enterocolitis, probiotics and nondigestible food ingredients that stimulate the growth or activity of probiotics, e.g. prebiotics, reduced oxidative stress and inflammation as indicated by estimating cytokines and genes related to oxidative stress and TLR signaling, measured in the terminal ileum. However, under hyperoxic/hypoxic conditions, the protective effect of the supplemented food was much reduced; this result indicated the limits of the impact of probiotic therapeutics.108 In meta-analyses comparing probiotic-treated IBD cases occurring through October 1, 2011, with cases treated with a placebo and standard controls (no added treatment), Jonkers et al109 revealed an overall lower risk ratio for remission induction in active ulcerative colitis with VSL#3 than with Bifidobacterium-fermented milk.
Epithelial barrier function is a critical factor in homeostasis and in the induction of IBD. One possible mechanism through which probiotics exert their influence is by strengthening barrier function. Studies of epithelial cell lines have shown that Lactobacillus species can upregulate the adherence and phosphorylation of junctional proteins β-catenin and E-cadherin, thereby enhancing barrier function—at least in vitro.110 Intestinal barrier maturation in mice takes place within the first 3 weeks of life. Recently, it has been demonstrated that colonization by commensal bacteria and interactions of these microorganisms with the innate immune system, using antibiotic treatment and MYD88 deficiency as tools of investigation, are critical for the expression of claudin-3, a tight-junction protein.111 Enteral administration of live or heat-killed L. rhamnosus GG induces similar claudin-3 expression, but the live product also exacerbates mortality risk. The latter study shows that live bacteria can cause discrepant outcomes and that heat-killed bacteria can have a probiotic impact without the added harmful effects. Accordingly, a lysate of L. casei was protective in a model of dextran sulfate sodium (DSS)–induced colitis.112 In another study, heat-killed Lactobacillus brevis SBC8803 enhanced barrier function and other immunomodulatory effects; as a result, the survival rate increased among mice with DSS-induced colitis.113 However, no increase in antimicrobial peptides or tight-junction proteins was seen when the impact of L. paracasei was studied in an adoptive transfer model of colitis.114 Instead, protection was associated with reduced neutrophil accumulation and pro-inflammatory cytokine expression (e.g., expression of IL-1β, IL-6, and IL-12).
Increased Treg function is another mechanism usually associated with probiotic efficacy. When supplemented with low numbers of Tregs, SCID mice tube-fed with L. acidophilus NCFM or L. salivarius Ls-33 for 5 weeks were protected against CD4+CD25− adoptive transfer–induced colitis.115 However, the reduced pro-inflammatory cytokine levels in the sera of these mice were not indicative of protection conferred by Tregs: In the absence of these regulatory cells, probiotic-treated mice had reduced serum levels of these cytokines. Thus, protection by the probiotic was judged to be based on the promotion of a Treg-favorable environment rather than on a direct effect on Tregs.
One mechanism of suppressing DSS-induced colitis is the activation of the aryl hydrocarbon pathway receptor. Lactobacillus bulgaricus OLL1181 activated messenger RNA expression of cytochrome P450 family 1A1 (CYP1A1)—a target gene of the aryl hydrocarbon pathway receptor pathway—in human colonic cells in vitro and in murine colonic cells in the DSS-induced colitis model; the experimental mice were protected from disease.116
Besides the administration of “good bacteria” to patients, another approach is the manipulation of dietary nutrients to create a microenvironment in which these bacteria can thrive.117 Future investigations into IBD etiology and individualized therapeutic interventions may rely on an understanding of complex gene–environment interactions. The impact of diet can be different or similar in ulcerative colitis and Crohn's disease. Foods rich in monosaccharides or disaccharides and total fats but low in micronutrients (especially vitamin D) increase the risk of both diseases, whereas high-level intake of vegetables and fruits and high-level intake of dietary fiber lowers the risk of ulcerative colitis and Crohn's disease, respectively.118 Compared with probiotic bacteria, prebiotics, such as galacto-oligosaccharides, pose less cumbersome storage challenges and can be used in wide range of “designer foods” as supplements.119
Oral delivery of genetically transformed L. lactis bacteria producing cathelin-related antimicrobial peptide (mCRAMP) alleviated clinical symptoms and mucosal damage in a DSS-induced colitis model; in contrast, sulfasalazine reduced clinical symptoms but not mucosal damage.120 These results provide a classic example of the advantages of biological therapy over that with comparatively ineffective drugs. Neutralization of reactive oxygen species, which are involved in intestinal inflammation, can offer another pathway to restoration of homeostasis. When given to mice before and after intrarectal administration of TNBS, L. casei BL23 bacteria engineered to produce either catalase or superoxide dismutase protected the animals more efficiently than the native bacterial species.121 Surprisingly, an inherent probiotic effect of this bacterial species was not observed: Mice receiving bacteria that were not engineered had disease scores equal to those in mice that did not receive bacterial supplementation.
Identifying and isolating immunomodulatory molecules from members of the commensal microbiota seem to represent the final frontier in beneficial exploitation of “our friends within.” DNA from commensal bacteria is pro-inflammatory limiting Treg conversion.122 However, to control IBD, commensal molecules must also possess the opposite—i.e., anti-inflammatory—properties. An example of a commensal molecule with potent anti-inflammatory activity is lactocepin, an enzyme from L. paracasei.123 This protease selectively degrades IFN-γ–inducible protein 10 (IP-10), a lymphocyte-recruiting chemokine, thus reducing inflammation. Lycogen, an extract from the anaerobic bacterium Rhodobacter sphaeroides, exhibits anti-inflammatory properties in the DSS-induced colitis model.124 Compared with untreated diseased mice, Balb/c mice orally treated with lycogen had reduced histopathological abnormalities, decreased pro-inflammatory cytokine expression, and increased survival rates. These effects of lycogen may be directly related to its modulation of macrophage activity, which was documented in an earlier study by the same group. The peptide STp from Lactobacillus plantarum was found to induce the anti-inflammatory cytokine IL-10 in intestinal DCs from healthy donors. In addition, DCs pulsed with this peptide induced an immunoregulatory cytokine profile and skin-homing profile in stimulated T cells.125 Peptides like STp hold promise as novel therapeutic and diagnostic tools for use against IBD pathologies.
Mechanism of Action of a Prototypical Commensal Microbial Molecule
PSA, a zwitterionic polysaccharide from the capsule of the human gut commensal B. fragilis, possesses immunomodulatory properties that influence both arms of the immune system. PSA interacts with the innate immune system through TLR2, primarily on antigen-presenting cells but also on CD4+ T cells.15,126 After endocytosis by antigen-presenting cells and processing by nitric oxide, PSA is presented to CD4+ T cells on major histocompatibility class II molecules.127,128 These interactions result in activation of CD4+ T cells, an event that has both IFN-γ–induced pro-inflammatory consequences (e.g., maturation of the host immune system) and IL-10–induced anti-inflammatory consequences (e.g., protection from inflammation-induced pathologies both in the gut and in distant tissues).129 Recently, we observed that control of inflammation-induced pathologies relies critically on PDCs (S. Dasgupta et al, unpublished data, 2013). In the presence of PSA, PDCs are potentiated to induce IL-10 production by CD4+ T cells. This potentiation is dependent on TLR2 and co-stimulator expression on the PDCs. In depletion and adoptive transfer experiments in a murine model of colonic inflammation, PDCs are essential for the immunoprotective activities of PSA. Finally, in a murine model of multiple sclerosis, we observed high mortality rates in groups of mice treated with a PDC-depleting antibody, irrespective of PSA treatment; in contrast, isotype control–treated mice survived and were protected by PSA treatment.
CONCLUSIONS AND FUTURE DIRECTIONS
The commensal microbiota can serve as a natural aid to mammalian mechanisms of defense against pathogenic and inflammatory insults. This microbial community has co-evolved in a host-specific manner, imprinting functionality on the host with regard to immune maturation.130 It is reasonable to think of exploiting the functional repertoire of the microbiota in ways that benefit human health—for example, by enhancing the clinical management IBD. Patients tend to be cautiously interested in the use of probiotics, which they generally consider a potential natural alternative to immunosuppressive drugs.131 However, given that the Internet often provides misleading or downright incorrect information, physicians must clearly inform their patients about the source and type of natural therapy and its realistic potential.132,133
In addition to investigating the treatment of IBD specifically, it is important to pursue knowledge of the role of the commensal microbiota in general. Inflammation has an immense influence on other disease processes, including cancer; thus, a relation between the commensal microbiota and colorectal cancer is conceivable. Recently, Arthur et al134 demonstrated an invasive carcinoma exacerbation due to monocolonization of azoxymethane-treated, spontaneously colitic IL-10 KO mice with E. coli NC101. This tumor-enhancing activity was localized to a genotoxic island (polyketide synthase) in the E. coli gene. The clinical relevance of this study became clear when increased numbers of E. coli bacteria bearing mucosa-associated polyketide synthase were found in patients with IBD and in colorectal cancer patients.
Further information about the correlation of changes in the microbiota with particular physiological states in the host will certainly open up a path to future innovative research. For example, an important influence of the microbiome during pregnancy has been reported.135 The third-trimester microbiota differed vastly from the first-trimester commensal community—notably, in including increased numbers of Proteobacteria and Actinobacteria. Stool samples from women in the third trimester yielded indications of inflammation and, when transferred to germ-free mice, induced greater adiposity and insulin insensitivity than did first-trimester stool.
One caveat applies to future investigations of the impact of the gut microbiota on the mammalian host's health: Researchers must take care not to overemphasize its significance. Although effects of gut commensalism have been recognized in distant tissues, the local microbiota may, in fact, play a predominant role in controlling immunity at these sites. For example, immunity to skin pathogens is mediated by the local microbiota.136 Nevertheless, considering the overall profound effect of the gut microbiota and the fact that the density of commensal bacteria is maximum in the gut, investigations to exploit this natural resource is paramount to the betterment of patients in future.
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