Christina Grosserichter Wagener

The immune system provides continuous protection for the human body against infections and outgrowth of aberrant cells. This requires a tight balance, especially at mucosal surfaces of the respiratory and intestinal tract to prevent outgrowth of pathogens, and at the same time to support colonization by harmless commensal bacteria. The challenge for the immune system is to tolerate the harmless bacteria, while clearing pathogens. Innate immune cells, such as monocytes, dendritic cells, neutrophils and macrophages, respond quickly without much specificity for the involved pathogens to control the balance between tolerance vs. clearance. In contrast, the adaptive immune responses, formed by B and T cells, take longer, are highly specific and characterized by memory formation. B cells recognizing antigen (foreign material) with their B-cell receptor, become activated, proliferate and differentiate into memory B cells and antibody (immunoglobulin; Ig)-producing plasma cells. T cells are activated by small protein fragments (peptides) presented in the context of the major histocompatibility complex (MHC) by antigen presenting cells, such as dendritic cells. Subsequently, T cells proliferate and differentiate into memory T cells and effector cells. Dendritic cells produce soluble molecules, which can direct the immune system, e.g. in the mucosa, they can direct B cells to produce IgA antibodies and induce the differentiation of regulatory T cells.

In and on mucosal surfaces of our respiratory and intestinal tracts, IgA is the main antibody produced. The highest bacterial load is found in the intestine, which contains high amounts of bacteria that are essential for the breakdown of food products. The exact composition of the intestinal bacterial microbiota has long been unknown because the majority of species cannot be cultured. However, this has been overcome over the past 20 years with the development of techniques for the massively parallel analysis of the RNA and DNA sequence (sequence analysis). The introduction of fast sequence analysis of the conserved 16S rRNA gene of bacteria enabled the identification of small differences between bacterial species and strains to identify the exact composition of the intestinal microbiota. Associations between alterations of intestinal bacterial composition and immune mediated diseases, such as rheumatoid arthritis, diabetes, atopic diseases or inflammatory bowel disease, have been described. While intestinal bacteria are presumed to affect the adaptive immune system and thereby disease susceptibility, these effects have been understudied.

In the first two chapters of my thesis, I present the results of our investigations into whether the adaptive immune response can be linked to distinct intestinal microbiota composition. We characterized intestinal bacteria from feces samples through sequence analysis of the 16S RNA gene, and in parallel performed phenotyping of blood T and B-cell subsets by 11-color flow cytometry. Adults (Chapter 2) and children (Chapter 3) were clustered into three enterotypes defined by similar intestinal microbiota composition. Two enterotypes were similar between adults and children, one enterotype was characterized by a high relative abundance of the genus Bacteroides and the other enterotype was characterized by a high relative abundance of Prevotella. The third enterotype was different and was defined in adults by high relative abundance of Ruminococcaceae and in children by high abundance of Blautia. In adults, we showed that reactivity of IgA from plasma and intestines towards various commensal and opportunistic bacteria is similar in individuals with different enterotypes. In general, we found in plasma higher IgA reactivity towards bacteria than in IgA isolated from feces. The immunophenotyping showed that adults with the Ruminococcaceae enterotype had reduced T-helper cell numbers of the type Th17, Th17.1 and Th22 in blood. In children with the Blautia enterotype, we found higher CD4+ effector memory T cells, CD27- IgA+ and CD27+ IgE- memory B cells in blood. These studies showed that individuals with distinct intestinal microbiota differ in composition of their B- and T-cell compartments. Due to differences in the third enterotype between adults and children, further studies are needed to confirm our results. These studies could investigate whether bacterial metabolites, such as short chain fatty acids, play a role, and what the effect of these on the function of cells is, such as cytokine production.

In addition to healthy individuals, we also studied the IgA response in immunological diseases. Patients with selective IgA deficiency (sIgAD) have a substantial decrease (<0.07 g/l) or even lack of IgA antibodies in blood. The majority of people with sIgAD have no clinical symptoms; however, about 30% of them suffer from recurrent infections, allergies, autoimmune diseases or can develop common variable immunodeficiency (CVID). The study described in Chapter 5 focuses on symptomatic patients. By studying the molecular maturation, perform B-cell stimulation and study IgA memory B cells in blood, we showed that IgA RNA contained similar somatic hypermutation levels as in healthy controls, and that in-vitro B-cell activation was also normal. However, T-cell dependent and T-cell independent circulating IgA memory B cells were decreased, as were Th1 and Th17 T cells. The concentrations of soluble molecules BAFF, APRIL and TGF-β1 in blood were increased. These increased cytokine concentrations and decreased T-helper cell numbers are suggestive of a B-cell extrinsic defect, and a disturbance in the regulation of the IgA response. Future studies should further elaborate our results to obtain more insights in the ethology of sIgAD and the clinical representation of the disease. An overreaction to harmless environmental particles at mucosal surfaces takes place in atopic diseases (i.e. IgE mediated allergies). We studied healthy children and children with atopic dermatitis, inhalant or food allergies participating in the Generation R Study (Chapter 4). We performed detailed phenotyping of T and B cells in blood of 10-year-old children. Children with any atopic disease had more regulatory T cells, Th2 cells, Th17 cells, naive B cells and IgA memory B cells, whereas IgE memory B cells were normal. Specifically, children with food allergies had increased naive B cells, CD27+ IgA+ memory B cells, regulatory T cells and Th2+ cells. These results show differences in the IgA response can also be found in atopic disease. Further studies should elaborate on the role of TGF-β in induction of regulatory T cells and IgA memory B cells in individuals with food-allergies. In conclusion, in this thesis we show that children and adults with distinct enterotypes show differences in their blood memory B- and T-cell compartments. These insights can guide further studies to investigate the immune modulating properties of the intestinal microbiota. In selective IgA deficiency, extrinsic defect(s) affect the regulation of the IgA response and can be involved in the etiology of the disease. More studies to understand the regulation of the IgA response will improve the understanding of the pathophysiology of selective IgA deficiency and increase the knowledge about the role of IgA in other diseases, such as IgE-mediated allergies.