Age-Related Alteration of Arginase Activity Impacts on Severity of Leishmaniasis

It is well documented that ageing alters many aspects of immune responses; however, a causal relation between impaired immune functions in ageing individuals and the response to infection has not been established. Experimental leishmaniasis is an excellent model to analyse protective and pathological immune responses. Leishmania parasites are obligate intracellular pathogens and invade mainly macrophages, which have dual function: they can kill the parasites or promote their growth. We have recently shown that arginase, an enzyme induced in infected macrophages, is a key factor for parasite survival. Here, we show that ageing reduces the expression levels of arginase in macrophages, resulting in more efficient control of parasite growth. Our results suggest that age-related differences in the metabolism of arginase in macrophages might contribute to the higher susceptibility of children to leishmaniasis

Local Suppression of T Cell Responses by Arginase-Induced L-Arginine Depletion in Nonhealing Leishmaniasis

The balance between T helper (Th) 1 and Th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized Th1 or Th2 responses are not sufficient to account for healing or nonhealing. Here we show that high arginase activity, a hallmark of nonhealing disease, is primarily expressed locally at the site of pathology. The high arginase activity causes local depletion of L-arginine, which impairs the capacity of T cells in the lesion to proliferate and to produce interferon-γ, while T cells in the local draining lymph nodes respond normally. Healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. Moreover, competitive inhibition of arginase as well as supplementation with L-arginine restored T cell effector functions and reduced pathology and parasite growth at the site of lesions. These results demonstrate that in nonhealing leishmaniasis, arginase-induced L-arginine depletion results in impaired T cell responses. Our results identify a novel mechanism in leishmaniasis that contributes to the failure to heal persistent lesions and suggest new approaches to therapy

Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice

Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs). Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid. The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasisThe study was supported in Spain by grants from Ministerio de Ciencia e Innovación FIS PI11/00095 and FISPI14/00366 from the Instituto de Salud Carlos III within the Network of TropicalDiseases Research (VI P I+D+I 2008-2011, ISCIII -Subdirección General de Redes y Centros de Investigación Cooperativa (RD12/0018/0009)). This work was also supported in Brazil by a grant from CNPq (Ciencia sem Fronteiras-PVE 300174/2014-4). A CBMSO institutional grant from Fundación Ramón Areces is also acknowledged. EAFC is a grant recipient of CNPq. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Intestinal Organoids in Inflammatory Bowel Diseases

Ulcerative colitis (UC) and Crohn’s disease (CD) are the two major forms of inflammatory bowel diseases (IBD), which are chronic and progressive diseases of the gastrointestinal tract. The exact cause is unknown, but it has been suggested that the diseases result from an abnormal immune response towards environmental factors, such as the microbiota, in a genetically predisposed person. Current therapies aim to stop this inflammatory cascade. The most widely used and successful therapy so far has been anti-tumour necrosis factor (TNF) antibodies, but also drugs with other modes of action are currently in use in the clinic (anti-adhesion molecules) or will soon become available (anti-interleukin (IL-) 12/23 and anti-Janus kinase inhibitors). Many patients are primary non-responders, and another large group of patients develop secondary loss of response over time to these antibodies. Discovering new possible therapeutic targets is therefore of great importance, which is still difficult due to insufficient knowledge of disease pathogenesis. Microbiota not only play an important part in disease, but also in homeostasis. A dysbiosis in the microbiota has been described in IBD, with an increase in pro-inflammatory species and a lack of anti-inflammatory bacteria. Furthermore, patients with IBD may have decreased mucosal barrier function, resulting in an influx of bacteria towards and in the epithelium. The epithelium consists of crypts and villi, with absence of villi in the colon. All intestinal epithelial cells are generated by Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5)+ intestinal stem cells (ISCs), which are located at the bottom of the crypts. ISCs undergo differentiation when they move upward due to cellular drift away from the niche, which provides stem cell factors. In IBD, due to leakiness of the barrier, an interaction may take place between bacteria and ISCs, but little is known about the effect microbiota may have on ISC functioning. Organoids are an excellent model to study the specific interaction between ISCs and microbiota. Intestinal organoids consist out of ISCs, and grow as self-organizing 3D structures in Matrigel, which constitutes the extracellular matrix. With factors as Wnt family member 3A (WNT3A), R-spondin-1, noggin, epidermal growth factor and others, cells undergo long-term expansion while remaining genetically stable. In this project, we investigated if organoids may be used in IBD research, as a future model for organoid-microbiota interactions. First, we established a library of intestinal organoids from patients with IBD and control patients without IBD, by isolating crypts from mucosal biopsies and growing them as organoids. We found no statistically significant differences in organoid forming capacity between controls and UC or CD patients. However, we observed a non-significant trend of decreased organoid forming efficiency of biopsies derived from macroscopically inflamed areas. To see if organoids retain all biological functions, we cultured established organoids for at least two weeks, and then performed a differentiation by withdrawal of WNT3A, nicotinamide and p38-inhibitor. Under these conditions, ISCs differentiate in all the downstream cell lineages found in the intestinal epithelium. We found that in differentiated organoids, MUCIN2 mRNA levels (a goblet cell marker) were decreased in CD organoids compared to controls. Furthermore, Atonal bHLH Transcription Factor 1 (ATOH1), a transcription factor controlling differentiation of cells to the secretory lineage, was expressed at significantly lower levels in differentiated organoids from both UC and CD patients vs. controls. There was a significant increase in differentiated organoids from UC patients vs. controls for Tight junction protein 1 (TJP1), and a trend for increased expression in CD organoids. This increase in TJP1 is in direct contrast with the increased permeability seen in patients with IBD. It may be that there is a decrease of other tight junction proteins and perhaps these organoids are compensating by up-regulating TJP1. Lastly, when comparing ileum and colonic organoids we noticed, as reported before, that organoids retain their location specificity, as reflected by increased levels of villin-1, alkaline phosphatase (enterocyte marker), and chromogranin A (enteroendocrine cell marker) in ileal samples. Due to heterogeneity in differentiation, we were unable to corroborate the found differences on mRNA levels on the protein level. Next, we aimed to investigate if organoids have an inflammatory response when challenged with inflammatory stimuli, such as TNFα, or the bacterial compounds muramyl dipeptide (MDP) and lipopolysaccharide (LPS). LPS nor MDP evoked a response in transcriptional activity after 24 hours exposure to three different concentrations of LPS or MDP (0.5 µg/ml, 5 µg/ml, and 10 µg/ml), while 100 ng/ml TNFα induced expression of various genes: C-X-C motif chemokine ligand- (CXCL) 8 and 3, TNFα, Nuclear factor kappa B (NFκB) and two NFκB inhibitors. Our results were in line with previously reported findings that gastric organoids also are unresponsive to LPS, but do express CXCL8 after exposure to flagellin, TNFα, or live Helicobacter pylori culture. Patients with IBD experience disease flares followed by disease remission. How mRNA levels of inflammatory markers develop over time, from primary tissue to organoids, was unknown. We therefore included patients with active inflammation (macroscopically assed during endoscopy). We obtained normal and inflamed appearing tissue from the same patient, performed crypt isolation and organoid culture, and analysed mRNA expression levels in biopsies and organoids after 14 days in culture. As expected, LGR5 levels were enriched in organoids vs. biopsies. Markers of inflammation, IL1β and TNFα, were generally increased in UC and CD patients’ biopsies. The expression of these markers was decreased in organoids in all patient groups compared to the original biopsies. Strikingly, we observed an increase in CXCL8 for all groups in organoids vs. controls, suggesting that culture conditions induce its expression or it is required for organoid growth. CXCL3 expression levels were comparable to CXCL8 trends. Importantly, overall expression levels in organoids between the different patient groups were not significantly altered. We also performed this for ileal tissues from CD patients and controls, and found mostly similar patterns in de- or increases in the analysed genes. Thus, at the mRNA level, markers of inflammation reach baseline levels in organoids quickly, but it needs to be proven whether this is due to removal from the (inflammatory) environment, or that non-epithelial cells (e.g. immune cells) are responsible for the detected inflammation in biopsies. Although the organoid model is very suitable to determine the effect of microbiota or its components on ISCs, it does not include other cell types such as those from the immune system. We therefore also performed in vivo experiments using a murine model of colitis, in which mice received 2.5% dextran sodium sulphate (DSS) in drinking water for 5 days, followed by 7 days of recovery with normal drinking water. It had been shown previously that colitis leads to the ablation of LGR5+ ISCs in a reporter mouse model (LGR5-LacZ) after 5 days of DSS, and upon withdrawal of DSS it takes 3-5 days before these cells reappear, indicating that there is regeneration through another source of cells. Our data were in line with what had been reported previously. Lgr5 expression levels were still at only 50% 7 days after withdrawal of DSS. Musashi RNA-binding protein 1 (Msi1), a marker of reserve ISCs, was equally affected as Lgr5. On the level of inflammation, we found that expression of TNFα peaked directly after 5 days of DSS, and it was not significantly altered compared to control levels 7 days after withdrawal of DSS. Interferon gamma (IFNγ) and IL1β on the other hand increased after 5 days of DSS with the highest increase 7 days after withdrawal of DSS, indicative of a primary trigger by TNFα, but a more TH-driven immune response later. Furthermore, IL1β may also have a function in epithelial recovery. This model may be used to study in vivo effects of microbiota on colitis and ISCs. In conclusion, organoids derived from IBD patients show largely comparable transcriptional profiles to control patient organoids. Furthermore, organoids derived from normal and inflamed tissues quickly reach baseline mRNA levels, and unexpectedly, CXCL3 and CXCL8 were induced in organoids. We could confirm previously published findings of the effect of colitis on ISCs in a murine model, and this will facilitate studies on the influences of microbiota. We have established a large organoid library from controls and patients with IBD, which represent a reliable in vitro model for further studies and characterization of the response of organoids with different genetic backgrounds to microbial or other stimuli.Acknowledgements iii Summary vi Samenvatting ix List of abbreviations xv Chapter 1: Introduction 1 1.1. Inflammatory bowel diseases 3 1.1.1. Epidemiology 4 1.1.2. Genetics 5 1.1.3. Environment 8 1.1.4. Microbiota 10 1.1.5. Involvement of the immune system 13 1.1.6. The intestinal epithelium 16 1.2. Intestinal epithelial models 19 1.2.1. 2D models 19 1.2.2. 3D models 20 1.2.3. Disease modelling with organoids 22 1.2.4. Dissecting the effect of microbes on the intestinal epithelium 23 1.2.5. Comparison of models 24 1.2.6. Mouse models of inflammatory bowel diseases 27 Chapter 2: Research objectives 31 Chapter 3: Establishing organoid cultures from patients with IBD and controls 35 3.1. Introduction 37 3.2. Materials and Methods 37 3.3. Results 43 3.4. Discussion 55 Chapter 4: Characterization of organoids from patients with IBD and controls 58 4.1. Introduction 60 4.2. Materials and Methods 60 4.3. Results 64 4.4. Discussion 76 Chapter 5: Inflammation in intestinal organoids 82 5.1. Introduction 84 5.2. Materials and Methods 84 5.3. Results 86 5.4. Discussion 96 Chapter 6: The effect of dextran sodium sulphate induced colitis on murine intestinal stem cells 99 6.1. Introduction 101 6.2. Materials & methods 102 6.3. Results 104 6.4. Discussion 108 Chapter 7: General discussion and future perspectives 113 References 127 Curriculum vitae 149 Personal Contribution 151 Conflicts of Interest Statements 152nrpages: 170status: publishe

Epithelial organoid cultures from patients with ulcerative colitis and Crohn's disease: a truly long-term model to study the molecular basis for inflammatory bowel disease?

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Human intestinal epithelium in a dish: Current models for research into gastrointestinal pathophysiology

Determining the exact pathogenesis of chronic gastrointestinal diseases remains difficult due to the complex in vivo environment. In this review we give an overview of the available epithelial cell culture systems developed to investigate pathophysiology of gastrointestinal diseases. Traditionally used two-dimensional (2D) immortalised (tumour) cell lines survive long-term, but are not genetically stable nor represent any human in particular. In contrast, primary cultures are patient unique, but short-lived. Three-dimensional (3D) organoid cultures resemble the crypt-villus domain and contain all cell lineages, are long-lived and genetically stable. Unfortunately, manipulation of the 3D organoid system is more challenging. Combining the 3D and 2D technologies may overcome limitations and offer the formation of monolayers on permeable membranes or flow-chambers. Determining the right model to use will depend on the pathology of interest and the focus of the research, defining which cell types need to be included in the model.status: publishe

Helicobacter pylori γ-glutamyl transpeptidase and vacuolating cytotoxin promote gastric persistence and immune tolerance

Infection with the gastric bacterial pathogen Helicobacter pylori is typically contracted in early childhood and often persists for decades. The immunomodulatory properties of H. pylori that allow it to colonize humans persistently are believed to also account for H. pylori's protective effects against allergic and chronic inflammatory diseases. H. pylori infection efficiently reprograms dendritic cells (DCs) toward a tolerogenic phenotype and induces regulatory T cells (Tregs) with highly suppressive activity in models of allergen-induced asthma. We show here that two H. pylori virulence determinants, the γ-glutamyl transpeptidase GGT and the vacuolating cytotoxin VacA, contribute critically and nonredundantly to H. pylori's tolerizing effects on murine DCs in vitro and in vivo. The tolerance-promoting effects of both factors are independent of their described suppressive activity on T cells. Isogenic H. pylori mutants lacking either GGT or VacA are incapable of preventing LPS-induced DC maturation and fail to drive DC tolerization as assessed by induction of Treg properties in cocultured naive T cells. The Δggt and ΔvacA mutants colonize mice at significantly reduced levels, induce stronger T-helper 1 (Th1) and T-helper 17 (Th17) responses, and/or trigger more severe gastric pathology. Both factors promote the efficient induction of Tregs in vivo, and VacA is required to prevent allergen-induced asthma. The defects of the Δggt mutant in vitro and in vivo are phenocopied by pharmacological inhibition of the transpeptidase activity of GGT in all readouts. In conclusion, our results reveal the molecular players and mechanistic basis for H. pylori-induced immunomodulation, promoting persistent infection and conferring protection against allergic asthma

Biopsy-derived intestinal epithelial cell cultures for pathway based stratification of patients with inflammatory bowel disease

Endoplasmic reticulum stress was shown to be pivotal in the pathogenesis of inflammatory bowel disease. Despite progress in IBD drug development, not more than one third of patients achieve steroid-free remission and mucosal healing with current therapies. Furthermore, patient stratification tools for therapy selection are lacking. We aimed to identify and quantify epithelial ER stress in a patient-specific manner in an attempt towards personalized therapy.status: publishe

Development of organoids from mouse and human endometrium showing endometrial epithelium physiology and long-term expandability

The endometrium, which is of crucial importance for reproduction, undergoes dynamic cyclic tissue remodeling. Knowledge of its molecular and cellular regulation is poor, primarily owing to a lack of study models. Here, we have established a novel and promising organoid model from both mouse and human endometrium. Dissociated endometrial tissue, embedded in Matrigel under WNT-activating conditions, swiftly formed organoid structures that showed long-term expansion capacity, and reproduced the molecular and histological phenotype of the tissue's epithelium. The supplemented WNT level determined the type of mouse endometrial organoids obtained: high WNT yielded cystic organoids displaying a more differentiated phenotype than the dense organoids obtained in low WNT. The organoids phenocopied physiological responses of endometrial epithelium to hormones, including increased cell proliferation under estrogen and maturation upon progesterone. Moreover, the human endometrial organoids replicated the menstrual cycle under hormonal treatment at both the morpho-histological and molecular levels. Together, we established an organoid culture system for endometrium, reproducing tissue epithelium physiology and allowing long-term expansion. This novel model provides a powerful tool for studying mechanisms underlying the biology as well as the pathology of this key reproductive orga