Towards novel biomarkers and rational nutritional interventions in Inflammatory Bowel
Disease
von Martels, Julius Zweder Hubertus
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Publication date: 2019
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von Martels, J. Z. H. (2019). Towards novel biomarkers and rational nutritional interventions in Inflammatory Bowel Disease. Rijksuniversiteit Groningen.
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GENERAL INTRODUCTION
AND OUTLINE OF THIS THESIS
GENERAL INTRODUCTION
Inflammatory Bowel Disease (IBD) predominantly comprises Crohn’s disease (CD) and Ulcerative colitis (UC). CD has an increasing incidence; and it currently affects more than two million people in Europe.1 The peak age of onset is in late adolescence or early
adult-hood, with no apparent difference between males and females.1
Clinically, exacerbations of CD are typically characterized by symptoms such as abdom-inal pain, diarrhea, fatigue and weight loss. However, symptoms may in some cases be aspecific, and there is often a delay between onset of the disease and the date of diag-nosis. Postponed medical treatment can negatively affect the disease course in these patients, and these patients more often require intestinal surgery than patients that are diagnosed and treated without delay.2 According to the European Crohn’s and Colitis
Organisation (ECCO) consensus of 2010, the diagnosis of CD is confirmed by clinical eval-uation and a combination of endoscopic, histological, radiological and/or biochemical in-vestigations.3 Endoscopically, CD is typically characterized by discontinuous involvement,
(peri-)anal lesions and a cobblestone appearance. The terminal ileum (45%), colon (32%) or ileocolonic segment (19%) are the most frequently affected locations of disease in CD but can also affect the upper gastrointestinal tract.4
CD has a multifactorial and complex disease origin. Environmental factors, the genetic predisposition of the individual and the composition of the intestinal microbiota all inter-act and contribute to development of the disease, and underlie the inappropriate intesti-nal immune reaction in these patients. 5 The Western lifestyle and diet, high in processed
foods (with large amounts of fat and refined sugar) and low in dietary fiber, has been implicated to contribute to the growing incidence of this disease in the industrialized countries. 6 Other, well-known environmental risk factors for CD include cigarette
smok-ing and the use of non-steroidal anti-inflammatory drugs (NSAIDs). 7, 8 Moreover, in recent
years, an increasing number of genetic loci have been identified to predispose for the development of CD.9 Finally, dysbiosis of the gut microbiota is a well-described feature in
CD. Below, we will elaborate on the dysbiosis that is frequently observed in CD patients, and potential strategies to restore this dysbiotic state.
The gut microbiome composition in CD is characterized by an increase in the number of pathogenic bacteria and a reduction in beneficial bacteria. Moreover, a decrease in the diversity of commensal bacterial species is described. 10 In CD, there is a reduction in
the abundance of multiple butyrate-producing bacteria species. Butyrate belongs to the Short-Chain Fatty acids (SCFAs) and is known to have anti-inflammatory and
anti-carcino-genic effects on the gut epithelium. 10, 11 The obligate anaerobic Faecalibacterium
praus-nitzii, is known to be a major butyrate-producer in the human gut. 12 Many studies show
that F. prausnitzii is reduced in abundance in CD patients. 13, 14 F. prausnitzii has shown
to have different anti-inflammatory properties. 15, 16 Moreover, the microbiota composition
of CD patients might predict the course of the disease to some degree; and in this way function as a biomarker for progress of the disease. 17 In addition, this patient-specific
microbiota composition (or ‘microbiota signature’) might predict response to therapy. 18
This can contribute to identifying patients with a high risk of a severe disease course; and guide the physician to an improved selection of appropriate medical treatment (‘‘per-sonalized medicine’’).
In vitro laboratory models of the gut can help to investigate the interaction between the gut microbiota and the intestinal/gut epithelium at a mechanistic level. These mod-els might be helpful in understanding the basis of dysbiosis and the consequence for host-microbe interactions in IBD. Also, these models may allow for testing food ingredi-ents that might be instrumental in correcting dysbiosis. In recent years, many different laboratory models for the gut have been established. In Part I of this thesis, we give an overview of recently developed aerobic-anaerobic co-culture systems. Particularly, we will focus on the ‘Human oxygen bacteria anaerobic’ (HoxBan) co-culture system. This co-culture system allows us to study the interaction between an obligate anaerobic bac-teria, such as F. prausnitzii and oxygen-requiring intestinal epithelial cells in vitro. Currently, endoscopic examination is one of the few reliable means to diagnose the dis-ease course of IBD patients. Less invasive means to diagnose IBD inflammatory status might lead to earlier intervention and better management of the disease and might pre-vent surgery. In Part II of this thesis, we focus on the role of inflammatory biomarkers and intestinal permeability in IBD. The correlation between patient-reported symptoms such as the Harvey-Bradshaw index (HBI) or the Crohn’s Disease Activity Index (CDAI) and endoscopic disease activity is suboptimal in CD. 19, 20 Prolonged subclinical inflammatory
disease activity entails an increased risk of the development of disease complications and requirement for surgical therapy. Novel non-invasive biomarkers that can predict intestinal inflammation in an early stage are warranted. 21, 22 Novel techniques enable us
to quantify the concentrations of inflammatory biomarkers (serum cytokines, chemokines and markers for angiogenesis and vascular injury) in a highly sensitive and validated man-ner. It is an interesting research objective to compare the predictive value of established inflammatory markers such as clinical disease indices, CRP, and fecal calprotectin to the predictive value of these inflammatory biomarkers. Moreover, establishing a correlation between the mucosal status as determined with endoscopic evaluation and
concentra-tions of these inflammatory biomarkers has a large potential to increase predictive accu-racy of inflammatory disease activity.
During increased inflammatory activity in IBD, oxidative stress, characterized by an in-creased production of reactive oxygen species (ROS), plays an important role in the pathogenesis of CD. 23, 24, 25, 26 Systemic oxidative stress is associated with reduced plasma
free thiol levels (R-SH, sulfhydryl groups), since these thiols are rapidly oxidized by reac-tive oxygen metabolites. 27 The role of free thiols has been evaluated in several
(inflam-matory) disease conditions, however, until now, its role in CD has not been investigated but might be a novel essential target to manage IBD and serve as biomarker for disease severity. 28, 29, 30
Permeability of the intestine might also be an essential biomarker for disease progress. Enhanced intestinal permeability (IP) in IBD and increased inflammatory activity has been established. 31, 32 Currently, orally administered tracers for IP play no significant role in
monitoring inflammatory activity in CD. Numerous studies have assessed the role of
51Cr-labeled ethylenediaminetetraacetic acid (51Cr-EDTA), however, its role has not been
sufficiently established, due to relatively small and heterogeneous IBD study cohorts. 33, 34, 35, 36, 37, 38, 39, 40 Recently, a non-radioactive and preferred alternative has been developed,
in which the 52Cr isotope has been incorporated to 52Cr-EDTA. It would be of interest to
evaluate the role of this novel 52Cr-EDTA test in a large cohort of CD patients, and to
compare the chromium excretion between CD patients with normal and increased inflam-matory disease activity (i.e. normal and increased feces calprotectin levels). Moreover, a possible link between chromium excretion and the Montreal disease classification and a link with key bacterial marker strains (F. prausnitizii and Enterobacteriaceae) of CD dys-biosis has not been explored in detail before.
Currently the treatment goal of IBD consist of the induction and maintenance of remission. In many cases maintenance medication is required. Currently prescribed medications are targeted on reducing or altering the immune response and consist of 5-aminosalicylates, glucocorticoid drugs, immunomodulators (e.g. azathioprine and methotrexate) and bio-logical antibodies against cytokines or surface markers on inflammatory cells (e.g. inflix-imab, adalimumab, ustekinumab, vedolizumab etc). Additionally, certain studies indicate that nutrition may have a therapeutic role in IBD. As important illustrative example, exclu-sive enteral nutrition has been shown to induce remisexclu-sive disease in children with CD. 41, 42 When taking into account the knowledge gained on biomarkers and disease
progres-sion we designed a dietary intervention in CD patients in Part III of this thesis. This was done with riboflavin (vitamin B2) which has been suggested to increase the abundance
of F. prausnitzii in the gut, because of riboflavin’s role in the anaerobic respiration of this bacterium (extracellular electron shuttling). 43 Also, riboflavin has several antioxidant and
anti-inflammatory effects and might via this mechanism reduce symptoms of IBD. 44, 45 46 To
determine whether riboflavin can influence F. prausnitzii abundance in IBD a pilot study was designed in healthy individuals. This study demonstrated that a short period of sup-plementation of the diet with riboflavin (2 weeks 100 mg per day) increased the relative abundance of F. prausnitzii in the feces in this short term supplementation period. 47 The
results led to the design of a larger intervention study in which the effect of administration of a riboflavin supplement in a cohort of CD patients was evaluated.
OVERALL AIM OF THIS THESIS
This thesis consists of the above described three parts. The overall aim of this thesis is to further unravel the complex interaction between diet, the gut microbiota and the epitheli-um in CD patients. Additionally, this thesis is focused on identifying novel biomarkers (se-rum inflammatory biomarkers, and biomarkers for intestinal permeability and systemic ox-idative stress) for predicting inflammatory disease activity in a minimally invasive manner.
OUTLINE OF THIS THESIS
PART I
Laboratory models that focus on the interaction between the anaerobic gut bacteria and oxygen-requiring gut epithelial cells are relevant for improved understanding of gut dis-eases. In Chapter 2, we give an overview of recently developed co-culture systems that allow research of host-microbe interactions involving anaerobic bacteria. These systems might lead to fundamental understanding of host-microbe interactions in health and dis-ease such as in IBD and it allows for testing efficacy of food components to correct dys-functional interactions between host-cells and microbes.
In Chapter 3, we describe the effect of F. prausnitzii in the ‘Human oxygen bacteria anaerobic’ (HoxBan) co-culture system. The interactions between the strict anaerobic bacteria F. prausnitzii and the gut epithelial cells are analyzed in this model.
PART II
CD is a chronic disease that often follows a relapse-remitting disease course. There are a number of biomarkers used to early detect an exacerbation of CD. In clinical practice, fecal calprotectin is the most commonly used non-invasive biomarker. 48 In Chapter 4,
we quantify a number of relevant cytokines, chemokines and markers for angiogenesis and vascular injury and correlate their concentrations to fecal calprotectin levels in a well described CD cohort.
In a follow-up study, described in Chapter 5, we measured an array of 10 relevant in-flammatory biomarkers in a larger IBD cohort. Furthermore, in a subset of this cohort, we determined the mucosal status with endoscopic evaluation. The degree of mucosal in-flammation, as quantified with either the SES-CD (CD) or the Mayo endoscopic subscore (UC) was represented by a composite IBD endoscopy score, that was correlated to our subset of inflammatory biomarkers.
In Chapter 6, we investigate the adequacy of systemic redox status in CD patients as biomarker for disease progress. To this end a comparison of plasma free thiols in CD patients is performed with healthy individuals. Plasma free thiols accurately reflect sys-temic redox status and have potential antioxidant effects, and for this reason may serve as biomarker for disease progress but might also serve as a potential target for therapy. Within this CD cohort, we study putative associations between plasma free thiols and established disease parameters, especially markers of inflammatory disease activity.
Barrier dysfunction might also serve as biomarker for disease progress as enhanced permeability is a hallmark of active CD. The intestinal epithelial cells form an important barrier between the external environment (luminal content) and the host cells. This bar-rier function may be compromised in IBD. In Chapter 7, we investigate the place of the
52Cr-EDTA oral permeability test in CD in relation to several disease parameters, including
fecal calprotectin levels, CD disease location as defined by the Montreal classification and relative abundance of key bacterial marker strains of CD (F. prausnitzii and Entero-bacteriaceae).
PART III
In Chapter 8, we tested the effect of riboflavin supplementation on multiple biomarkers and outcomes of disease in CD patients (results of the RISE-UP clinical trial). An additional reason to test riboflavin is that it hypothetically enhances beneficial microbes such as F. prausnitzii which typically is reduced in abundance in the gut of CD patients when com-pared to healthy individuals. 14 We hypothesize that this supplement might reduce
symp-toms of CD by either reducing oxidant stress or by increasing the relative abundance of this bacterium. We evaluate the effect on CD disease-specific symptoms (Harvey-Brad-shaw Index, HBI) and Quality of Life (QoL).
Finally, in Chapter 9, we summarize the main findings of the this thesis. In addition, we propose several suggestions for future research.
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