Gut microbiome-intestinal barrier crosstalk during Iron deficiency anaemia: use of nutrition-based Strategies

Abstract

Anaemia is a multifactorial syndrome characterised by a reduction in haemoglobin levels below homeostatic ranges, which vary according to age, gender and physiological conditions. It is estimated to affect one third of the global population with an increased prevalence in underdeveloped countries, where it imposes a considerable economic burden. Iron deficiency is the top leading cause of anaemia, causing approximately 50% of cases, and the most prevalent micronutrient deficiency worldwide. The lack of variety in therapeutic alternatives is one of the major problems when treating iron deficiency anaemia (IDA). Oral iron (iron supplements) is the first treatment option, with side effects outnumbering its beneficial aspects. Since iron absorption is fairly limited, non-absorbed iron from supplements is accumulated in the intestine, triggering oxidative stress and damaging the intestinal epithelium. Excess of iron also causes alterations in the gut microbiome. Not surprisingly, gastrointestinal alterations are common side effects produced by oral iron supplements, which in the end lead to the abandonment of treatment before iron deposits have been completely restored andto repetitive episodes of IDA. A considerable percentage of iron deficiency anaemias become chronic and refractory to treatment. Although it is well known that iron supplements exert a detrimental effect on gut health, questions regarding the alterations in the gut microbiome and the state of the intestinal epithelium and the gut barrier during IDA remain unanswered. The human intestine harbours the greatest and most diverse microbial community in the entire organism and causal relationships have been repeatedly established between the gut microbiome and the state of health. Some studies have investigated the microbial alterations in the context of IDA using classical methods and suggest that microorganisms might contribute to IDA through competition for iron with the host or by altering intestinal homeostasis. Most of these studies have focused on the microbial communities in the large intestine and faeces and did not use state-of-the-art sequencing methods. The gut microbiome is a key component of the intestinal barrier and intestinal dysbiosis has often been associated with alterations in the gut barrier. In physiological conditions, there is a wide variety of antigens, toxins and microorganisms in the intestinal lumen, and the gut barrier aims to prevent their leakage into the bloodstream. There is a considerable body of evidence to suggest that an increased leakage to extraintestinal tissues can trigger inflammation and lead to the aggravation of certain disorders. Despite the fact that iron shortage can impair DNA replication and cell cycle progression, therefore impairing the gut barrier, this aspect has been barely studied during IDA. Since addressing the deterioration of the gut microbiome and the intestinal barrier, if altered during IDA, would be one of the goals when optimising IDA therapies, fermented goat’s milk (FGM) was studied in this thesis as a nutritional tool to restore gut health. The higher nutritional value of goat’s milk along with its reduced risk of allergy compared to cow’s milk make it suitable to be used in specific scenarios. One of the nutrients showing the highest prebiotic potential are oligosaccharides, whose concentration is higher in goat’s milk compared to other animals. They are more structurally diverse and bear a close resemblance to breast milk oligosaccharides, the golden standard in relation to beneficial effects on intestinal health. On the other hand, fermentation improves digestibility as well as sensory properties and nutritional value of milk, increasing the availability of oligosaccharides and enhancing their prebiotic impact. Goat’s milk has been shown to increase iron bioavailability, but whether this effect might be microbiome-dependent is not known. Goat’s milk oligosaccharides have been reported to exert beneficial effects on the gut microbiome and the gut barrier integrity, but studies describing the effect of FGM are scarce. Therefore, a full characterisation of the alterations occurring in the gut microbiome along the gastrointestinal tract during IDA together with an in-depth analysis of the intestinal barrier have been included as the two hallmarks of intestinal health to study in this thesis. Moreover, the IDA recovering capacity of FGM was studied, along with its gut microbiome-shaping properties and its effects on the intestinal barrier. IDA was experimentally induced in an animal model through the use of an ironfree diet (iron free AIN-93G diet) over a period of 40 days. At the end of the induction period, part of the animals were sacrificed. Blood, serum, intestinal content and intestinal mucous layer samples and faeces were collected from each intestinal region (duodenum, jejunum, ileum, cecum and colon). The remaining animals underwent the treatment period using either a FGM-based diet or a standard diet along 30 additional days. At the end of the treatment period, animals were sacrificed and similar samples were collected. After confirming IDA had been correctly induced through the determination of haematological parameters, the gut microbiome was analysed in intestinal contents belonging to all segments (duodenum, jejunum, ileum, cecum and colon) and in faeces. In a first approach, 16S rRNA sequencing (amplicon sequencing) was used to identify the region showing the greatest dysbiosis. Amplicon sequencing revealed an intestinal dysbiosis occurred in response to IDA, with intensity increasing towards the most distal segments of the digestive tract (large intestine: cecum and colon). Predictive microbial functional analysis on 16S rRNA sequencing data revealed that the most enriched microbial pathways in the large intestine during IDA were related to short chain fatty acid (SCFA) metabolism. Determination of SCFA (butyrate, propionate and acetate) confirmed an increase during IDA, especially in the colon. Taxonomic analysis revealed an enrichment in members of the genus Clostridium in the large intestine, one of the major SCFA producers, which showed high positive correlations to butyrate and propionate levels in the colon of anaemic animals. Therefore, the colon was identified as the region showing the greatest dysbiosis during IDA. To take an in-depth look at the gumicrobiome alterations, whole metagenome sequencing was applied on colonic contents, revealing an enrichment in Clostridium species during anaemia, positively correlated to butyrate and propionate levels. Beside SCFA-related pathways being more abundant during IDA, nucleotide-producing pathways and bacterial load were also higher in the anaemic animals. Hence, key microbial species belonging to the genus Clostridium and key metabolites were identified as the main drivers of changes in the gut microbiome occurring in response to IDA. Given their well documented positive role on the intestinal epithelium, SCFA are likely to be part of microbial trade-off mechanisms taking place during IDA to compensate for systemic or intestinal disease-derived alterations. Analysis of the intestinal barrier on the colonic mucous layer through mRNAsequencing revealed an underdeveloped intestinal epithelium during IDA due to the downregulation of Gene Onthology terms related to the development and synaptic signalling within the enteric nervous system, the development of the digestive tract, cell junction assembly and cell integrity and organization of the extracellular matrix. Since collagen metabolism, as the major component of the extracellular matrix, is iron dependent, the expression of collagen-related genes and proteins was specifically analysed, finding also decreased levels during IDA. Due to the well stablished role of SCFA on the stabilization of intestinal hypoxia and the impact of hypoxia on the maintenance of the intestinal barrier, hypoxia target genes were analysed by quantitative PCR, showing no differences in expression levels between the control and the anaemic group. Therefore, the observed alterations in the intestinal barrier were hypoxiaindependent. Microbial translocation was studied in serum samples using two biomarkers: lipopolysaccharide (LPS) and bacteria-specific immunoglobulins. Increased LPS translocation along with an increased immune response against dysbiotic bacteria is in accordance with an impaired intestinal barrier during IDA. During the treatment period, the determination of haematological parameters revealed IDA was more efficiently recovered using a FGM-based diet compared to a standard one. The gut microbiome-modulating properties of FGM were studied in intestinal content samples belonging to all intestinal segments and faeces collected after the treatment period. FGM showed microbiome-modulating properties in the small and large intestine of control animals in line with this increased efficiency. The gut microbiome shaped by the FGM-based diet was slightly more diverse in terms of the number of species in the microbial community and more functionally active than that shaped by the standard diet, which is confirmed by a higher bacterial load in the colon of animals fed with FGM-based diet. As far as its microbiome-restoring properties are concerned, FGM-based diet and standard diet similarly restored gut dysbiosis in the small intestine, since alterations in this region were less noticeable. Gut dysbiosis in the colon was more efficiently restored by FGM-based diet compared to standard diet, confirming its prebiotic potential. None of the diets completely restored the microbiome in relation to the control group during the employed treatment period. Lastly, FGM did not show any additional positive effect on the gut barrier compared to the standard diet. Both diets restored the altered expression levels of key downregulated genes during IDA involved in the maintenance of the intestinal barrier. Absence of differences in serum LPS concentrations between control and anaemic groups fed with FGM-based diet or standard diet suggest a similar state of the intestinal barrier after treatment with both diets. This thesis analyses the alterations in the gut microbiome and intestinal barrier occurring in an animal model that represents a nutritional iron deficiency. With the discovery of an intense gut dysbiosis in the large intestine with an apparent compensatory role and an overall deteriorated intestinal barrier that leads to an increased leakage of microbial components, taking gut-protective approaches seems imperative when treating IDA. Fermented goat’s milk was studied in this context, recovering IDA and restoring the colonic microbiome more efficiently. Despite the fact that fermented goat’s milk also restored the integrity of the intestinal barrier, no additional positive effects were shown in this regard in comparison with the standard diet. Fermented goat’s milk is therefore a useful nutritional tool to ease intestinal alterations occurring during iron deficiency and to reduce the negative impact of iron supplements on the gut health.