Accepted Manuscript

Exploring & exploiting our “other self” – does the microbiota hold the key to the future
therapy in Crohn’s?

Lea-Maxie Haag, MD Britta Siegmund, MD

PII: S1521-6918(14)00045-6
DOI: 10.1016/j.bpg.2014.04.001
Reference: YBEGA 1246

To appear in: Best Practice & Research Clinical Gastroenterology

Received Date: 29 January 2014

Revised Date: 9 March 2014
Accepted Date: 14 April 2014

Please cite this article as: Haag L-M, Siegmund B, Exploring & exploiting our “other self” – does
the microbiota hold the key to the future therapy in Crohn’s?, Best Practice & Research Clinical
Gastroenterology (2014), doi: 10.1016/j.bpg.2014.04.001.

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 ACCEPTED MANUSCRIPT
1 Exploring & exploiting our “other self” – does the microbiota hold the key to

2 the future therapy in Crohn’s?

4 Lea-Maxie Haag, Britta Siegmund*

5 Medical Department (Gastroenterology/Rheumatology/Infectious Diseases)

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6 Charité – Universitätsmedizin Berlin

7 Berlin, Germany

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8

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9

10 Lea-Maxie Haag, MD

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11 Medical Department (Gastroenterology/Rheumatology/Infectious Diseases)
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12 Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin

13 Hindenburgdamm 30, DE – 12200 Berlin (Germany)

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14 t: +49-30-450-514 342

f: +49-30-450 514 990

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16 Email: lea-maxie.haag@charite.de
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17
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18 Britta Siegmund, MD

19 Medical Department (Gastroenterology/Rheumatology/Infectious Diseases)

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20 Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin

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21 Hindenburgdamm 30, DE – 12200 Berlin (Germany)

22 t: +49-30-450 514 342

23 f: +49-30-450 514 990

24 Email: britta.siegmund@charite.de

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26 *Corresponding author
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1 ABSTRACT

2 Inflammatory bowel diseases (IBD) with its two major forms Crohn’s disease (CD)

3 and ulcerative colitis (UC) are chronic relapsing disorders leading to inflammation of

4 the gastrointestinal tract. Although the precise aetiology of IBD remains unclear,

5 several factors are believed to contribute to disease pathogenesis. Among these, the

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6 role of the intestinal microbiota has become more and more appreciated. Evidence

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7 from experimental and clinical studies strongly suggests that chronic intestinal

8 inflammation results from a dysregulated immune response towards components of

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9 the microbiota in genetically susceptible hosts. The growing perception of the

10 microbiota as a major driver of disease pathogenesis raises the question, if the

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intestinal microbiota can be used as a therapeutic target in CD. Based on what we
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12 know about host microbiota interactions in health and disease, the objective of this
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13 review is to address the question if the microbiota holds the key to the future therapy

14 in CD.
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18 Key words: Crohn’s disease, inflammatory bowel disease, microbiota, probiotics,

19 prebiotics
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1 Exploring and exploiting our “other self” – does the microbiota hold the key to

2 the future therapy in Crohn’s?

4 1. Introduction

5 The adult intestine contains more than 100 trillion microorganisms comprising of over

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6 1000 different species-level phylotypes thus making the mammalian intestine the

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7 largest and most complex component of the immune system. With its metabolic

8 activities and immune regulatory function the endogenous gastrointestinal microbiota

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9 displays a complex ecosystem, playing an important role in both normal physiology

10 and disease susceptibility (1). In health, our coexistence with the gut microbiota can

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be described as a dynamic, mutually beneficial relationship. Maintenance of this
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12 status requires balanced mechanisms regulating the host’s tolerance to the constant
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13 stimulus of the gut microbiota (2, 3).

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15 Inflammatory bowel diseases (IBD), encompassing Crohn’s disease (CD) and

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16 ulcerative colitis (UC), are chronic relapsing inflammatory conditions affecting the

17 gastrointestinal tract. Although the aetiology of these diseases remains unclear,

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18 several factors are believed to play a role in its development and progression

19 including host genotype, dysregulated immune responses and the composition of the
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commensal intestinal microbiota (4). Evidence from genetic, immunologic and

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21 microbial studies implicates that chronic intestinal inflammation results from a

22 dysregulated immune response towards components of the commensal intestinal

23 microbiota in genetically susceptible individuals (5). In the last two decades, the

24 perception of the microbial contribution to IBD has been diverted, shifting from a
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1 single infectious agent to the complex commensal intestinal flora as a major driver of

2 disease pathogenesis.

4 2. Intestinal microbiota

5 2.1 Composition and functions

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6 Composition of intestinal microbiota

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7 The human gastrointestinal tract harbours more than 100 trillion microbes, including

8 bacteria, archaea, eukarya and viruses (1). Our knowledge and understanding of the

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9 intestinal microbial composition was based on culture dependent techniques for

10 decades. Recent development of molecular, culture-independent profiling methods

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has broadened the view regarding microbial community composition (2). Culture
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12 independent, molecular techniques using 16s ribosomal RNA gene sequencing
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13 suggested that the human microbiota contains about 200 strains of bacteria

14 comprising of over 100 different bacterial species, and dominated by just a few phyla
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15 (6, 7). These techniques also revealed a high level of variability between individuals
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16 at the bacterial species level. Nevertheless, Firmicutes and Bacteroidetes are the

17 predominant phyla across all vertebrates, representing over 90% of all intestinal
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18 bacteria. Studies on the gut microbiota profiles of adult humans with varying degrees

19 of genetic relatedness revealed a high degree of similarity regarding microbiota

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composition in monocygotic twins, even though this composition remained distinct. In

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21 contrast, unrelated individuals living in the same environment, having the same

22 eating habits showed less similarity. These findings underline the crucial role of

23 genetic and environmental factors in gut microbiota development (8).

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1 Microbiota: host phenotype – genotype cross-talk

2 It has been estimated that the total number of genes from this complex microbiome

3 exceeds that of the human genome by approximately 100-fold (9). This coexistence,

4 the relationship between the host and the microbiota, has often been described as

5 commensalism, a disputable perception when considering the impact of microbiota

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6 on host gut physiology. The intestinal microbiota is substantial for mucosal barrier

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7 function, development of the mucosal immune system as well as for nutrient

8 processing including production of short-chain fatty acids (SCFA) and vitamins (10).

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9 The major amount of SCFA is derived from bacterial fermentation of undigested

10 carbohydrates. SCFA play a major role in the physiology of the colonic mucosa as

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they display a unique energy source for colonocytes (11). Conditions coming along
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12 with reduced SCFA-levels including diversion colitis, fibre-free diet or germ-free
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13 conditions result in metabolic starvation and consecutive colonic atrophy (12).

14 Recently, SCFA were shown to regulate the number and function of colonic
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15 regulatory T-cells thereby promoting colonic homeostasis. These findings underline

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16 the impact of SCFA on immune interactions between the intestinal mucosa and the

17 luminal microbiota (13). Intestinal bacteria metabolizing dietary fibre to SCFA mainly
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18 belong to the phylum Firmicutes such as Clostridium and Bifidobacterium species.

19 Gordon et al. could recently show an increased ratio of Firmicutes/Bacteroidetes in

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obese humans and genetically obese mice (14). Furthermore, colonization of

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21 germfree mice with an obese-microbiota induced weight gain, underlining the impact

22 of the intestinal microbiota on human energy metabolism beside the capacity of

23 transmissibility. Recent studies using nucleotide-binding oligomerisation domain 2

24 (Nod2)-deficient mice provided evidence for the concept that the genotype impacts

25 microbiota composition (15). Investigations on the microbiota of Nod2-deficient mice

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1 indicated an increased load and altered composition compared to their wild-type

2 counterparts. Moreover, analyzes of human samples carrying NOD2 variants also

3 revealed substantial changes in microbiota composition underlining the influence of

4 host genotype on its microbiota. Subsequent studies showed that the substantial

5 changes in the microbiota composition caused by Nod2 deficiency is paralleled by an

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6 increased colitis severity following chemically induced injury (16). Remarkably, co-

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7 housing and cross-feeding experiments revealed the transmissibility of the observed

8 genotype-dependent disease risk. These studies allow for establishing the concept

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9 that the microbiota determines the host phenotype and vice versa the host genotype

10 dictates the microbiota composition.

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12 Microbiota – role for integrity and development of the mucosal immune system
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13 The intestinal microbiota plays a crucial role in host immune system development

14 and maintenance of the intestinal epithelial barrier. Murine models of germ-free

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15 animals revealed numerous developmental defects including fewer and smaller

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16 Peyer’s patches and mesenteric lymph nodes (MLN) as well as a thinner lamina

17 propria, fewer lymphoid follicles and plasma cells in germinal centres and a marked
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18 reduction of antibody production (i.e. IgA) compared to their conventionally raised

19 counterparts. In addition, normal functioning of intestinal epithelial cells including

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expression of microbial recognition receptors, defensins and antimicrobial peptides

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21 was shown to be impaired in germfree animals (17). Recent data from germfree mice

22 indicate that the time point of intestinal colonization is fundamental for the “balanced”

23 development of immune cell populations thus protecting the host from intestinal

24 inflammation (18).

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1 Investigations on segmented filamentous bacteria (SFB) revealed that this member

2 of the commensal microbiota is required for the induction of T helper (Th) 17 cells in

3 the small intestinal lamina propria (19). Th17 cells play a crucial role in protecting the

4 host from bacterial and fungal infections by secretion of IL-17, IL-17F and IL-22,

5 which in turn improve cell tight junction and antimicrobial protein production. Anyhow,

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6 one should not disregard that Th17 cells also have an inflammatory potential as they

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7 have been identified as key mediators of autoimmune diseases (20, 21). Recently,

8 certain strains within Clostridia clusters XIVa, IV and XVIII were shown to affect

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9 regulatory T-cell differentiation, accumulation and function in the murine colon.

10 Moreover, Atarashi and colleagues could show that affection of the Clostridium load

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in neonatal SPF mice by oral inoculation resulted in resistance to dextran sodium
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12 sulphate (DSS)-colitis in these mice in adulthood (22, 23).
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14 Prospectively, the ultimate aim will be to define an educative bacterial cocktail to be

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15 applied earlier in life thus allowing for a balanced development of the intestinal
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16 integrity. However, at this point further studies are required to achieve this goal.

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18 3. Intestinal microbiota in the pathogenesis of IBD

19 3.1 Experimental evidence

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Experimental studies of spontaneous or induced intestinal inflammation provide

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21 strong evidence that antigens derived from the intestinal microbiota drive chronic

22 colitis and ileitis, thereby playing a major role in initiating intestinal inflammation.

23 About two decades ago, IL-2-deficient mice were shown to spontaneously develop

24 colitis when raised under specific-pathogen-free (SPF) conditions, whereas under

25 germ-free conditions the onset of colitis was delayed and the disease severity was
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1 mild compared to mice raised under SPF conditions (24). Similarly, Kühn and

2 colleagues observed that IL-10-deficient mice developed an attenuated disease with

3 regard to disease severity and expansion when kept in a facility with a defined

4 microbial environment (25). In addition, mice with a genetically altered T-cell receptor

5 and that resulted in a phenotype of spontaneous colitis failed to develop inflammation

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6 in the absence of a microbial environment (26). These striking observations were

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7 some of the first suggesting that the inflammatory response is triggered by enteric

8 bacteria.

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9

10 3.2 Clinical evidence

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As exemplarily referred above, several murine studies have highlighted the impact of
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12 the gut microbiota in intestinal inflammation development. In humans, CD is mainly
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13 observed in intestinal areas containing the highest concentrations of bacteria (i.e.

14 terminal ileum and colon). The first evidence for the involvement of the intestinal
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15 microbiota in disease pathogenesis came from clinical experiments. Diversion of

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16 faecal stream from a segment of the small intestine affected by inflammation led to

17 resolution of inflammation in CD patients. Furthermore, restoration of faecal stream

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18 and postoperative exposure of the neoterminal ileum to luminal contents induced

19 inflammation, indicating that the microbiota acts as a trigger in postoperative

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recurrence of CD (27). Numerous studies have demonstrated alterations in the

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21 microbiota composition as a common feature of CD. These detected changes are

22 also termed dysbiosis. The majority of studies revealed that CD patients show less

23 complex profiles of commensal intestinal bacteria and increased numbers of mucosa-

24 associated bacteria compared to healthy individuals. Alterations regarding microbial

25 diversity were mainly due to a decrease in Firmicutes and Bacteroidetes as well as

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1 increases in members of the Proteobacteria and Actinobacteria phyla (6). Reduced

2 numbers of certain members of Bacteroidetes such as Bacteroides fragilis (B. fragilis)

3 might contribute to a dysbalance of the mucosal integrity and thus be prone for

4 intestinal inflammation. This member of the commensal intestinal flora expresses

5 distinct surface polysaccharides. Polysaccharide A is the most immunodominant

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6 polysaccharide of B. fragilis and mediates the establishment of Th1/Th2 balance in

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7 the host (28). Among the members of the Firmicutes phyla, the species

8 Faecalibacterium prausnitzii (F. prausnitzii) has been shown to be less abundant in

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9 the intestinal microbiota of IBD patients. This butyrate-producing bacterium has well

10 documented anti-inflammatory potential. Sokol and colleagues provided evidence

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that a decrease in F. prausnitzii was associated with a higher risk of recurrence of
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12 ileal CD in patients after ileoceacal resection. Anti-inflammatory effects of the
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13 commensal bacterium were evaluated both in vitro and in vivo. Stimulation of

14 peripheral blood mononuclear cells by F. prausnitzii resulted in significantly lower

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15 amounts of IL-12 and IFNγ and higher secretion of IL-10, underlining its anti-
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16 inflammatory properties. Oral administration of either live F. prausnitzii or its

17 supernatant reduced the severity of inflammation induced by 2,4,6-

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18 trinitrobenzenesulphonic acid in an in vivo colitis model (29, 30).

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20 In conjunction with the detected decreases in members of the Bacteroidetes and

21 Firmicutes phyla, there have been several reports regarding the increase of members

22 associated with the Proteobacteria and Actinobacteria phyla. For example, CD

23 patients were shown to contain an increased abundance of Enterobacteriaceae in

24 their faecal samples and mucosa-associated microbiota (31). Given these dysbiotical

25 changes, a ratio of F. prausnitzii / E. coli was suggested to serve for evaluation of the
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1 level of existing dysbiosis to identify patients with a high risk for disease recurrence

2 (32).

4 Metabolic consequences of the altered microbial composition and subsequent

5 changes in the intestinal environment derive from the multitude of their essential

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6 functions outlined above. Noteworthy, it is still not clear if there is a significant

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7 difference regarding dysbiosis in active and inactive disease. Likewise, it is still up for

8 discussion if dysbiosis is a cause or consequence of intestinal inflammation.

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9 Interestingly, analyses of faecal samples from asymptomatic first-degree relatives of

10 CD patients revealed a different microbiota composition compared with controls

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derived from families without CD history (33). These findings suggest a condition of
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12 “predysbiosis” preceding disease manifestation. Further evidence for an aetiological
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13 role of dysbiosis in IBD is provided by the transmissibility of inflammation

14 demonstrated in different in vivo-models as discussed above. However, there are

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15 also numerous findings supporting the theory of dysbiosis as a consequence of the

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16 inflammatory milieu like similar patterns of microbial changes in widely diverse hosts

17 in the presence of inflammation. Longitudinal studies may determine whether a

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18 particular microbiota profile can serve as a diagnostic tool to identify people carrying

19 a greater risk for developing IBD.

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21 Of note, indirect evidence from genome-wide association studies (GWAS) strongly

22 supports an involvement of the intestinal microbiota in CD pathogenesis. The large

23 majority of identified genetic variants in IBD patients are shared by CD and UC.

24 Different studies have identified CD-specific genes involved in innate immunity and

25 autophagy including NOD2 and autophagy-related protein 16-like 1 (ATG16L1). In

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1 this context it has been proposed that inappropriate immune responses to luminal

2 bacteria and thereby defective processing of intracellular bacteria could contribute to

3 disease development in genetically susceptible hosts (34, 35).

4 Maintenance of intestinal homeostasis depends on dynamic and reciprocal

5 interactions among the epithelium, the immune system and the intestinal microbiota.

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6 The aforementioned microbiota functions, host-microbiota interactions and genetic

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7 aspects underline the association of the intestinal microbiota with key aspects of host

8 physiology and immune homeostasis. The better understanding of the interplay

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9 between microbiota and the host is the prerequisite for the attempt to evaluate and

10 potentially harness these species as a possible therapeutic target in inflammatory

11 disorders such as CD.

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13 4. Potential for manipulation

14 Probiotics
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15 Probiotics are defined as living microorganisms which, when administered in

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16 adequate concentration, exert benefits on human health. Genera frequently

17 associated with probiotic activity include Lactobacillus, Bifidobacterium as well as

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18 lactic acid-producing bacteria such as Lactococcus and Streptococcus. Other

19 probiotics include organisms of the genera Bacillus, Bacteroides, Enterococcus,

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Faecalibacterium and nonbacterial organisms such as Saccharomyces boulardii (36).

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21 Several in vitro-studies revealed their mechanisms of suppressing inflammation. For

22 example, Lactobacillus johnsonii (L. johnsonii) induced transforming growth factor-β

23 (TGFβ) production in leucocyte-sensitised intestinal epithelial cell lines (37). VSL#3, a

24 preparation containing eight different lactic acid-producing bacteria, as well as its

25 three included Bifidobacterium species were shown to induce IL-10 production by

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1 human dendritic cells (DC), whereas its component Lactobacillus and Streptococcus

2 species failed at inducing IL-10 (38). Next to induction of anti-inflammatory cytokines

3 such as TGFβ and IL-10, several studies revealed the ability of probiotics to stimulate

4 the proliferation of regulatory T-cells (39). Further experimental evidence for

5 beneficial effects of probiotics derive from numerous in vivo studies using

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6 experimental models of intestinal inflammation (exemplarily illustrated in Table 1).

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7 The beneficial effects of probiotics are thought to arise through multiple mechanisms

8 including modulation of barrier functions, alteration of gut microbial diversity as well

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9 as the modulation of innate and adaptive immune effector mechanisms (17, 29, 37,

10 40-51).

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12 Table 1
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14 Given the promising data from both in vitro- and in vivo-studies, the question arises
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15 whether these observations can be translated into clinical practice. The evidence for
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16 probiotics in pouchitis therapy is persuasive. There are a couple of small trials

17 showing benefits of probiotic treatment in UC. For example, VSL#3 was

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18 demonstrated to be effective in induction of remission in a meta-analysis including

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19 three controlled trials (52). Different studies have demonstrated similar efficacy of
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20 mesalamine and E. coli Nissle 1917 in maintenance therapy of UC (53-55).

21 Accordingly, guidelines recommend probiotic therapy in distinct situations of UC and

22 pouchitis (56). There are only few studies investigating the efficacy of probiotics in

23 patients with CD and results obtained from these studies are conflicting (36, 57).

24 Studies addressing the role of probiotics for induction of remission in CD are almost

25 non-existent. A study investigating the use of probiotics in active CD derives from

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1 paediatrics and represents rather a case summary. Here the beneficial effects of

2 Lactobacillus rhamnosus GG (LGG) regarding disease activity and intestinal

3 permeability were assessed in four patients with active CD despite concomitant

4 immunosuppressive therapy (58). Colonization with LGG was accompanied with

5 sustained reduction of disease activity, associated with improved intestinal

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6 permeability in all patients. While promising, no solid studies were performed to

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7 confirm these preliminary findings. Thus to date, the therapeutic effect of probiotics in

8 active CD has not been sufficiently assessed.

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9

10 The use of probiotics for maintenance of remission (achieved either by medication or

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curative resection) was studied in a few trials as outlined in Table 2. Evidence for the
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12 use of probiotics as maintenance therapy in CD is not persuasive. Although there
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13 was a trend towards minor relapse rates in the probiotic group in some trials,

14 differences were not significant in most cases and only two studies reported positive
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15 results. Guslandi and colleagues evaluated the role of S. boulardii in maintenance

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16 treatment of CD (59). 32 patients with medically induced remission were randomized

17 to receive either S. boulardii (1 g) plus mesalamine (2 g) or mesalamine (3 g) alone.

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18 Clinical relapses assessed by CD activity index (CDAI) were observed in 37.5% of

19 patients treated with mesalamine alone and in 6.25% of patients treated with
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mesalamine plus the probiotic agent. In one study, treatment with the antibiotic
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21 rifaximin for nine months followed by VSL#3 for another three months resulted in

22 reduced percentage of post-operative relapses compared to 12-month treatment with

23 mesalamine (60).

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1 In addition, there were three studies evaluating the impact of LGG on maintenance of

2 remission (61-63). LGG was tested against placebo and no significant differences

3 were found with regard to clinical or endoscopic recurrence and time to relapse.

4 Finally, two randomised controlled double blind trials have reported no effect of

5 L. johnsonii in preventing post-operative recurrence (64, 65).

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6

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7 A randomized, double blind trial including 28 patients receiving prednisolone

8 investigated the efficacy of supplementary E. coli Nissle versus placebo in

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9 maintenance of remission (66). Even though remission rates were comparable in

10 both groups, there were some minor effects. All patients randomized to receive E.

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coli Nissle along with prednisolone had stopped the steroids within six months after
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12 entering the trial, while a few patients randomized to receive placebo could not stop
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13 their steroid intake. Still, the similar remission rate in both groups strongly suggests

14 that the remission was mostly attributable to steroid use.

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16 A recently conducted randomized controlled trial focussed on S. boulardii in relapse

17 prevention. The study enrolled 165 patients who had achieved remission after
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18 treatment with steroids or salicylates. After a study-period of 52 weeks, there were no

19 significant differences between the intervention and the placebo group pertaining to
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percentage of patients in remission and mean CDAI (67).

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22 Table 2

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1 Prebiotics & Synbiotics

2 Prebiotics include non-digestible short-chain carbohydrates such as fructo-

3 oligosaccharides (FOS) or galacto-oligosaccharides (GOS) or more complex

4 saccharides. The combination of probiotics and prebiotics is termed synbiotics (57).

5 Commensal bacteria are able to metabolize prebiotics thereby lowering luminal pH

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6 through their conversion to SCFA (68). Thus, prebiotics stimulate the growth and the

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7 metabolism of protective commensal microbiota. The efficacy of prebiotics is mostly

8 confined to in vitro studies and animal models of intestinal inflammation. Inulin,

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9 stimulating intracolonic SCFA production and modulating the composition of the

10 intestinal microbiota through an increase of lactic acid bacteria as well as FOS were

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shown to reduce the severity of experimental colitis (69, 70). There are only few
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12 human studies investigating the efficacy of prebiotics in CD including only a small
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13 number of patients. One of the first studies conducted, investigated the efficacy of

14 FOS in a small open label trial. The study assessed the effect of FOS administration
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15 in ten patients with active ileocolonic CD (71). Disease activity was assessed using
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16 Harvey Bradshaw index after daily administration of 15 mg FOS for 3 weeks.

17 Furthermore, concentrations of faecal and mucosal Bifidobacteria were quantified

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18 and mucosal DC IL-10 and Toll-like receptor (TLR) expression were assessed. FOS

19 supplementation induced a significant decrease of disease activity index

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accompanied with increased concentrations of fecal Bifidobacteria as well as a

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21 significant increase in TLR4 expressing DC. Recently, a large randomized, double-

22 blind, placebo-controlled trial failed in confirming these results (72). Another

23 randomized, placebo-controlled double blind trial investigated the effects of synbiotic

24 consumption (in addition to the current medication) on disease processes in patients

25 with active CD (73). The study involved 35 patients, the synbiotic comprised of
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1 Bifidobacterium longum and Synergy 1. Clinical status as well as histological scores

2 derived from rectal biopsies were assessed at initiation as well as after 3 and 6

3 months. The results indicated significant improvements in clinical outcomes as well

4 as histological scores in the synbiotic group. Yet another small study focussed on the

5 effect of probiotic and prebiotic co-therapy in active ileal, ilealcolonic or colonic CD

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6 (74). The ten patients enrolled had an initial therapeutic regimen consisting of

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7 aminosalicylates and prednisolon that failed to induce remission in these patients.

8 During the study period, all patients were on stable doses of aminosalicylates, home

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9 enteral nutrition and prednisolone (mean 8.5 ± 6.7mg qd). All patients received the

10 probiotic mixture (probiotics mainly comprised Bifidobacterium and Lactobacillus), six

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patients received an additional prebiotic psyllium dose. After a study period of 13
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12 months the authors reported a reduction in CDAI in seven patients. Six patients had
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13 a complete response (four of them received both pre- and probiotics), one patient of

14 the synbiotic group responded partially and three patients were identified as non-
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15 responders (2 of them only received probiotics). Two patients who received the
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16 additional synbiotic therapy were able to taper prednisolone, another four patients

17 (one of them belonging to the probiotics only group) were able to decrease its dose,
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18 whereas two patients receiving additional probiotics or the synbiotic respectively had

19 to increase their prednisolone. One has to underline the rather observational

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character of the study due to the limited number of patients included. Furthermore
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21 the results did not reveal any significant effect. In a recently conducted randomized,

22 double blind study Synbiotic 2000 was found ineffective in preventing postsurgical

23 recurrence in CD (75). Nevertheless, the role of an additional synbiotic strategy

24 remains to be determined.

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1 No relevant safety concerns were raised in any of the published studies thus it can

2 be stated that probiotic and prebiotic treatment of CD seems safe. CD – in contrast to

3 UC – appears to be totally resistant to pro- and prebiotic therapy. The few

4 aforementioned positive results underline the need for further research in this

5 therapeutic field. Interestingly, all trials claiming probiotic ineffectiveness in

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6 preventing disease relapse contained members of the genus Lactobacillus. In

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7 addition, in studies showing effectiveness of probiotics, Lactobacillus was not part of

8 the probiotic treatment. Therefore one could speculate that probiotics containing

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9 members of Lactobacillus alone are not capable of reducing risk of disease relapse.

10 Furthermore, dose-response studies are required to attribute the lack of response

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revealed in both pro- and prebiotic studies. It should also be considered that the
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12 responsiveness to a probiotic as well as prebiotic therapy depends on several
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13 variables including host variables (age, gender, lifestyle, compliance), risk factors

14 and disease distribution.

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16 Antibiotics

17 Antibiotics have an essential role in the treatment of complications of CD such as

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18 intra-abdominal and perianal abscesses as well as fistulae (76). Research studies

19 that demonstrate antibiotic efficacy in CD are still scarce and only a limited number of
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trials have evaluated broad-spectrum antibiotics in patients with active disease (3).
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21 More recently, studies in patients with CD revealed that rifaximine (800-1600 mg/d

22 for 2 weeks) reduced the number of treatment failure but did not facilitate the

23 induction of remission (77). Furthermore, antimicrobial therapy (clarithromycin

24 750 mg/d, rifabutin 450 mg/d and clofazimine 50 mg/d for 2 years) was shown to

25 significantly improve the short-term remission rate compared to controls (78).

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1 However, one has to consider that a long-term antibiotic treatment in the view of

2 resistance development is not desirable.

4 Faecal microbiota transplantation

5 Faecal microbiota transplantation (FMT) displays a new and underexplored

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6 therapeutic strategy to manipulate the intestinal microbiota. The use of human donor

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7 faeces as a therapeutic agent is established in serious cases of Clostridium difficile

8 (C. difficile) infections (79). Already in the late 1980s, FMT in a patient with UC

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9 resulted in durable clinical and histological cure (80). Until today, there is promising

10 evidence from case-reports, retrospective analyses as well as meta-analyses

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including small numbers of UC patients (81). There are currently several ongoing
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12 clinical studies to test the efficacy of FMT in CD patients. While FMT is intriguingly
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13 simple, the ultimate goal would be to define a selected composition of bacteria to be

14 administered luminally. However in view of the data defining the role of the
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15 microbiota on the development of the mucosal immune system as well as the divers
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16 effects induced by the different bacteria, defining a selected mixture might become

17 an experimental and clinical challenge.

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18

19 Summary
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Developing therapies for CD (and IBD in general) is particularly challenging due to its
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21 multifactorial aetiology. Based on numerous genetic, immunologic and microbial

22 studies, there is strong evidence for a microbial involvement in disease

23 pathogenesis. Considering the impact of the intestinal microbiota in experimental

24 disease pathogenesis, the observations in CD patients regarding microbial

25 composition and the results obtained from GWAS, it seems obvious to use the
 ACCEPTED MANUSCRIPT
1 microbiota as a therapeutic target in CD. Results obtained from probiotic and

2 prebiotic as well as antibiotic and FMT studies in CD patients are scarce. The few

3 studies showing a beneficial effect are less convincing and only included small

4 numbers of patients. However, these results require replication in higher-powered

5 studies as already proposed above. In conclusion, targeting the intestinal microbiota

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6 in the therapy of CD seems to be a promising concept but future research is required

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7 to reveal if this will become a generally accepted approach.

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 ACCEPTED MANUSCRIPT
Practice points

• Experimental and clinical evidence implicates that chronic intestinal

inflammation results from a dysregulated immune response towards

components of the commensal intestinal microbiota in genetically susceptible

individuals

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• genetic and environmental factors impact microbiota composition

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• the intestinal microbiota is capable of changing the host phenotype

• Studies using the intestinal microbiota as a therapeutic target in CD are

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scarce and show conflicting results

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1 AN
Research agenda

• Regarding the dysbiotical changes in CD patients, longitudinal studies are

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needed to determine whether a particular microbiota profile can serve as a

diagnostic tool to identify people carrying a greater risk for developing disease
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• Conflicting results from previous studies using the intestinal microbiota as

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therapeutic target underline the need for further research

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4
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5 Conflict of interest

6 none

7 Acknowledgement

8 The work was supported by the SFB 633, and SI 749/6-1 and 749/7-1 to B.S.

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Table 1 – Experimental evidence – probiotic effects in experimental intestinal inflammation
Animal model Probiotic compound / Mechanism of disease Ref
Bacterial strain suppression
Il10-/- VSL#3 colitis ↓; barrier ↑ 40
VSL#3 colitis ↓, inflammatory cytokines ↓ 41
VSL#3 regulation of IEC function 42
E. coli Nissle colitis ↓ 43
L. reuteri attenuation of inflammation, 44
bacterial translocation ↓
IFNγ ↓, IL-12p40 ↓

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L. plantarum 299v 45

TNBS-induced VSL#3 colitis ↓ (induction of IL-10 and 46

colitis TGFβ-expressing T cells)

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B. lactis colonic TNFα and iNOS ↓ 47
L. casei colonic cyclooxygenase 2 ↓ 47
L. salvarius colitis ↓ 48

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L. rhamnosus colitis ↓ 48
L. acidophilus colitis ↓ 48
L. casei colitis ↓ 48
colonic TNFα and iNOS ↓

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L. fermentum 47
L. fermentum colonic TNFα and iNOS ↓ 47
colitis ↓
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Faecalibacterium 29
prausnitzii

DSS-induced E. coli Nissle 1917 decreased colonic inflammation 49

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colitis via TLR2- and -4 dependent

pathways
E. coli Nissle 1917 colitis ↓ 50
Salmonella B. infantis induction of regulatory T-cells 51
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enterica induced
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VSL#3: preparation containing eight different lactic acid bacteria (Lactobacillus acidophilus, L. bulgaricus, L.
casei, L. plantarum, Streptococcus thermophilus, Bifidobacterium breve, B. infantis, B. longum);
DSS, dextran sulphate sodium; IFNγ, interferon-γ; IL, interleukin; iNOS, inducible nitric oxide synthase; TGFβ,
transforming growth factor-β; TLR, toll-like receptor; TNBS, trinitrobenzene sulphonic acid; IEC, intraepithelial
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cells; L., Lactobacillus; B., Bifidobacterium; E., Escherichia.

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Table 2. Clinical studies investigating probiotics in maintenance of remission in CD
Study groups Relapse rate %
Intervention Comparator n Desig Duration Interventio Comparato p Re
n (months n r f
)
S. boulardii Mesalamin 32 R, OL 6 6.25 37.5 0.0 59
plus e 4
mesalamin
e

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VSL#3 Mesalamin 40 R, OL 12 20 40 NR 60
e

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LGG Placebo 45 R, DB 12 17 (CR) 11 (CR) 0.3 61
60 (ER) 35 (ER) 61

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LGG Placebo 11 R, DB 6 50 60 NS 62

LGG Placebo 75 R, DB 24 31 17 0.1 63

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L. johnsonii Placebo 98 R, DB 6 49 64 0.1 64
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L. johnsonii Placebo 70 R, DB 3 15 (CR) 14 (CR) 0.9 65

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E. coli N Placebo 28 R, DB 12 86 92 NS 66
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S. boulardii Placebo 16 R, DB 52 47 53 NS 67
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S. boulardii, Saccharomyces boulardii; VSL#3: preparation containing eight different lactic acid bacteria
(Lactobacillus acidophilus, L. bulgaricus, L. casei, L. plantarum, Streptococcus thermophilus, Bifidobacterium
breve, B. infantis, B. longum); LGG, Lactobacillus rhamnosus GG; L. Johnsonii, Lactobacillus johnsonii; E. coli N,
E. coli Nissle; CR, clinical recurrence; ER, endoscopic recurrence; R, randomized; DB, double blind; OL, open
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label; NS, not significant; NR, not reported;

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