Butyrate producers as potential next-generation probiotics : safety assessment of the administration of Butyricicoccus pullicaecorum to healthy volunteers

Advances in gut microbiota research have triggered interest in developing colon butyrate producers as niche-specific next-generation probiotics, targeted at increasing colon butyrate production and countering disease-associated microbiota alterations. Crucial steps in the development of next-generation probiotics are the design of formulations with a reasonable shelf life as well as the safety demonstration of an intervention in healthy volunteers. One such potential next-generation butyrate-producing probiotic is Butyricicoccus pullicaecorum 25-3(T), with demonstrated safety in in vitro as well as animal models. Here, we examined the strain's safety, tolerability, and impact on microbiota composition and metabolic activity in healthy volunteers in a randomized, double-blind, placebo-controlled crossover study in 30 healthy volunteers. The study design consisted of two 4-week intervention periods (10(8) CFU B. pullicaecorum (treatment) or maltodextrin (placebo) per day) with a 3-week washout in between. We assessed adverse events, blood parameters (primary endpoints), and fecal microbiota composition and metabolite profiles (secondary endpoints). The number of reported adverse events during the B. pullicaecorum treatment was similar to that of placebo intervention, as were observed changes in blood chemistry parameters, bowel habits, and fecal calprotectin concentrations. Administration of the strain did not induce any disruptive effect in microbiota composition or metabolic activity. In this first human intervention trial with a butyrateproducing Clostridium cluster IV isolate, we demonstrated B. pullicaecorum 25-3(T) administration to be both safe and well tolerated by healthy participants. This safety study paves the way for the further development of the strain as a next-generation probiotic. IMPORTANCE This study is the first to determine the safety and tolerance in humans of a butyrate-producing Clostridium cluster IV next-generation probiotic. Advances in gut microbiota research have triggered interest in developing colon butyrate producers as next-generation probiotics. Butyricicoccus pullicaecorum 25-3(T) is one such potential probiotic, with demonstrated safety in vitro as well as in animal models. Here, we produced an encapsulated B. pullicaecorum formulation that largely preserved its viability over an 8-month storage period at 4 degrees C. Administration of this formulation to healthy volunteers allowed us to establish the intervention as safe and well tolerated. The probiotic intervention did not cause disruptive alterations in the composition or metabolic activity of health-associated microbiota. The results presented pave the way for the exploration of the impact of the strain on microbiota alterations in a clinical setting

Contribution of the faecal metabolome and microbiome to the pathophysiology of ulcerative colitis

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) which is characterised by chronic, relapsing inflammation of the colonic mucosa. The most common clinical symptoms include bloody diarrhoea, abdominal pain, fatigue and weight loss. Conventional therapeutic options are mainly targeted at reducing the inflammatory process but are only partially successful and associated with a range of side effects. The exact patho-physiological processes underlying this disease remain elusive, although increasing evidence supports the involvement of aberrant immune responses to environmental and gut microbial triggers in genetically susceptible hosts. The colonic microbiota has an imbalanced composition in UC and has been suggested to play a key role in the initiation and progression of disease activity. Through bacterial fermentation, an extensive range of colonic luminal compounds are produced that are in close interaction with the colonic mucosa and might influence intestinal health. In the present PhD project we focused on the role of the colonic compounds in the pathophysiology of UC. Their composition was characterised in faecal samples from UC patients and compared with that from healthy controls. In addition, their capacity to affect various parameters of gut function was investigated and discriminating compounds were identified. Furthermore, the safety of a novel butyrate-producing strain was investigated in healthy subjects. In the first part of this project we showed that the induction of inflammation in an epithelial cell line downregulates the uptake and oxidation of butyrate, which is the main energy-providing process for colonocytes (Chapter 3). This confirms the hypothesis that the impaired colonic butyrate metabolism in UC patients is consequential to the inflammation. Simultaneous incubation with butyrate counteracted the reduced butyrate oxidation, indicating that increasing luminal butyrate levels might have therapeutic potential in UC. In the second part we characterised the microbial dysbiosis in UC by a reduced biodiversity, decreased abundances of butyrate-producing species and increased levels of some opportunistic pathogens such as Helicobacter and Escherichia/Shigella. Analysis of the faecal metabolome revealed an aberrant composition of colonic compounds compared to healthy controls, including decreased levels of the short-, medium- and long-chain fatty acids (SCFAs, MCFAs and LCFAs) whereas some amino acids and bile acids were increased in UC (Chapter 4). Subsequently, we investigated the functional impact of this microbial dysbiosis by evaluating whether the colonic compounds from UC patients influenced physiological processes relevant to the UC pathogenesis. Faecal water from UC patients reduced the gene expression of the first enzyme in the butyrate oxidation pathway in colonic cells (Chapter 5), provoked increased epithelial cytotoxicity (Chapter 6), decreased the epithelial barrier resistance (Chapter 7) and increased the production of inflammatory cytokines in human peripheral blood mononuclear cells (Chapter 8) in comparison to faecal water from HC. This indicates that the altered composition of colonic compounds in UC patients has a more pronounced detrimental effect on gut functioning compared to HC, suggesting they have the potential to directly trigger, sustain or aggravate intestinal inflammation in UC patients. Colonic compounds that were consistently associated with changes in these gut function parameters included SCFAs, MCFAs and LCFAs (beneficial) together with ethanol, dimethyl disulfide and amino acids (detrimental). Altering their luminal concentrations in UC, which can be achieved by dietary adjustments promoting carbohydrate fermentation, may be a promising strategy to improve the colonic health in UC. An alternative strategy to increase luminal butyrate concentrations and target microbial dysbiosis is the use of butyrate-producing bacteria. In the third part of this project we conducted a placebo-controlled cross-over exploratory phase 1 trial with daily administration of the butyrate-producer Butyricicoccus (B.) pullicaecorum 25-3T in 30 healthy subjects. The intervention proved to be safe and well-tolerated, paving the way for its use in clinical settings to test its therapeutic effects (Chapter 9). Furthermore, B. pullicaecorum intervention increased total microbial richness, indicating the promotion of a more stable and resilient intestinal ecosystem. In conclusion, the results obtained in this PhD add to the current understanding of the pathophysiology of UC. Microbial dysbiosis leads to an altered production of colonic compounds which adversely affect gut health in UC and may contribute to the initiation, propagation or aggravation of the inflammatory process. Dietary changes which promote carbohydrate over protein fermentation, resulting in increased microbial SCFA production, and the limitation of alcohol consumption may entail gut health benefits for UC patients.Dankwoord - Acknowledgements Table of contents List of abbreviations CHAPTER 1: General introduction 1.1 Ulcerative colitis 1.2 UC pathogenesis 1.3 Altered gut microbial metabolism in UC: a metabolomics overview CHAPTER 2: Research objectives PART I: Cytokine effects on butyrate metabolism CHAPTER 3: Inflammation-induced downregulation of butyrate uptake and oxidation is not caused by a reduced gene expression 3.1 Introduction 3.2 Materials and methods 3.3 Results 3.4 Discussion PART II: Colonic microbial metabolism in UC: Does it affect gut function? CHAPTER 4: Characterisation of faecal metabolite and microbiota profiles in ulcerative colitis 4.1 Introduction 4.2 Materials and methods 4.3 Results 4.4 Discussion CHAPTER 5: Impact of colonic compounds on butyrate metabolism in ulcerative colitis 5.1 Introduction 5.2 Materials and methods 5.3 Results 5.4 Discussion CHAPTER 6: Impact of colonic compounds on cytotoxicity in ulcerative colitis 6.1 Introduction 6.2 Materials and methods 6.3 Results 6.4 Discussion CHAPTER 7: Impact of colonic compounds on barrier function in ulcerative colitis 7.1 Introduction 7.2 Materials and methods 7.3 Results 7.4 Discussion CHAPTER 8: Impact of colonic compounds on inflammation induction in ulcerative colitis 8.1 Introduction 8.2 Materials and methods 8.3 Results 8.4 Discussion PART III: Safety of Butyricicoccus pullicaecorum in healthy subjects CHAPTER 9: Safety of the butyrate-producing strain Butyricicoccus pullicaecorum in healthy subjects: a placebo-controlled cross-over randomised intervention trial 9.1 Introduction 9.2 Materials and methods 9.3 Results 9.4 Discussion CHAPTER 10: General discussion and future perspectives 10.1 Colonic metabolome of UC patients is highly divergent 10.2 Colonic compounds consistently related to gut function 10.3 Importance of colonic microbial metabolism in the pathogenesis of UC 10.4 Translation of research findings into dietary practice 10.5 Limitations of the PhD project 10.6 Future perspectives CHAPTER 11: Summary - Samenvatting 11.1 Summary 11.2 Samenvatting Curriculum vitae List of publicationsnrpages: 248status: publishe

Butyrate Producers as Potential Next-Generation Probiotics: Safety Assessment of the Administration of Butyricicoccus pullicaecorum to Healthy Volunteers

Advances in gut microbiota research have triggered interest in developing colon butyrate producers as niche-specific next-generation probiotics, targeted at increasing colon butyrate production and countering disease-associated microbiota alterations. Crucial steps in the development of next-generation probiotics are the design of formulations with a reasonable shelf life as well as the safety demonstration of an intervention in healthy volunteers. One such potential next-generation butyrate-producing probiotic is Butyricicoccus pullicaecorum 25-3T, with demonstrated safety in in vitro as well as animal models. Here, we examined the strain's safety, tolerability, and impact on microbiota composition and metabolic activity in healthy volunteers in a randomized, double-blind, placebo-controlled crossover study in 30 healthy volunteers. The study design consisted of two 4-week intervention periods (108 CFU B. pullicaecorum [treatment] or maltodextrin [placebo] per day) with a 3-week washout in between. We assessed adverse events, blood parameters (primary endpoints), and fecal microbiota composition and metabolite profiles (secondary endpoints). The number of reported adverse events during the B. pullicaecorum treatment was similar to that of placebo intervention, as were observed changes in blood chemistry parameters, bowel habits, and fecal calprotectin concentrations. Administration of the strain did not induce any disruptive effect in microbiota composition or metabolic activity. In this first human intervention trial with a butyrate-producing Clostridium cluster IV isolate, we demonstrated B. pullicaecorum 25-3T administration to be both safe and well tolerated by healthy participants. This safety study paves the way for the further development of the strain as a next-generation probiotic. IMPORTANCE This study is the first to determine the safety and tolerance in humans of a butyrate-producing Clostridium cluster IV next-generation probiotic. Advances in gut microbiota research have triggered interest in developing colon butyrate producers as next-generation probiotics. Butyricicoccus pullicaecorum 25-3T is one such potential probiotic, with demonstrated safety in vitro as well as in animal models. Here, we produced an encapsulated B. pullicaecorum formulation that largely preserved its viability over an 8-month storage period at 4°C. Administration of this formulation to healthy volunteers allowed us to establish the intervention as safe and well tolerated. The probiotic intervention did not cause disruptive alterations in the composition or metabolic activity of health-associated microbiota. The results presented pave the way for the exploration of the impact of the strain on microbiota alterations in a clinical setting.status: publishe

Commonly used pancreatic Cre-driver lines secrete human growth hormone which profoundly alters islet phenotype

status: accepte

Impaired Islet Function in Commonly Used Transgenic Mouse Lines due to Human Growth Hormone Minigene Expression

The human growth hormone (hGH) minigene is frequently used in the derivation of transgenic mouse lines to enhance transgene expression. Although this minigene is present in the transgenes as a secondcistron, and thus not thought to be expressed, we found that three commonly used lines, Pdx1-Cre(Late), RIP-Cre, and MIP-GFP, each expressed significant amounts of hGH in pancreatic islets. Locally secreted hGH binds to prolactin receptors on β cells, activates STAT5 signaling, and induces pregnancy-like changes in gene expression, thereby augmenting pancreatic β cell mass and insulin content. In addition, islets of Pdx1-Cre(Late) mice have lower GLUT2 expression and reduced glucose-induced insulin release and are protected against the β cell toxin streptozotocin. These findings may be important when interpreting results obtained when these and other hGH minigene-containing transgenic mice are used.publisher: Elsevier articletitle: Impaired Islet Function in Commonly Used Transgenic Mouse Lines due to Human Growth Hormone Minigene Expression journaltitle: Cell Metabolism articlelink: http://dx.doi.org/10.1016/j.cmet.2014.11.004 content_type: article copyright: Copyright © 2014 Elsevier Inc. All rights reserved.status: publishe