Intestinal mucosal humoral response and neuro-immune interaction as contributors to the pathophysiology of diarrhea-predominant irritable bowel syndrome

Irritable Bowel Syndrome (IBS) is a chronic and prevalent gastrointestinal disorder which curses with intestinal motility alterations and abdominal pain. IBS constitutes a relapsing and potentially disabling disorder and, currently, there is no specific diagnosis biomarker and only palliative treatments are available. The absence of a well-established pathophysiology highlights the need of identifying the underlying organic causes of motility alterations and the onset of symptoms. In the intestinal mucosa of these patients a certain degree of inflammation has been identified together with an increased intestinal permeability and a higher activity of the immune response. Previous studies from our group showed an increased humoral activity in the intestinal mucosa of diarrhea-predominant IBS (IBS-D) patients, associated with more severity of the symptoms. IBS patients often present anxiety and depression, and dysfunction of the gut-brain axis features IBS onset and outcome. Considering intestinal mucosa is highly innervated and the existence of a bidirectional modulation between nervous and immune systems, the main objective of this thesis was to characterize the activation humoral response by neuro-immune mechanisms in the intestinal mucosa of IBS-D patients. To achieve our purpose, this project has been divided into three chapters. In chapter 1, we collected jejunal biopsies, blood and feces from IBS-D patients and healthy volunteers. We quantified immunoglobulins (Igs) in stool and observed higher levels of IgG in IBS-D group, more specifically IgG2 and IgG3, despite this last one did not reach statistical significance. The amount of total IgG positively correlated with the intensity of the abdominal pain reported by the patients. We conducted a phenotypical analysis in jejunal biopsies for the expression of CD38 and CD138 plasma cell marker, PGP9.5 neural marker and TACR1 (substance P receptor) expression, involved in nociceptive signaling. We observed that plasma cells and nerve endings are found in proximity and, when we performed a quantification of this distance by transmission electron microscopy (TEM), results showed plasma cells are significantly close to nerve endings in the IBS-D group. This distance inversely correlates with acute stress symptoms and depression score reported by patients, the later not reaching significance. In Chapter 2, an RNA-seq analysis was conducted in RNA extracted from IBS-D patients and healthy volunteer jejunal biopsies. We performed a Gene Set Enrichment Analysis with all the genes in these samples; we observed the humoral response immunological phenotype is enriched and 60% of the genes with a highest enrichment score are involved in Ig structure. Pathways associated to intestinal barrier function are also overrepresented in IBS-D. Finally, in Chapter 3, we evaluated several sources to obtain a B cell in vitro model to study the effect of neuropeptides on immune activity, more specifically substance P, in B cell activation/differentiation and Ig production. We confronted primary culture cells (B and plasma cells isolated from intestinal mucosa and blood) and an established B cell line (126BLCL). In vitro differentiated plasma cells obtained from blood were also analyzed. After conducting a deep phenotypic characterization, we concluded blood isolated B cells are the most suitable and feasible in vitro model for our purpose. The results of this thesis reinforce the hypothesis of the neuro-immune crosstalk in the intestinal as playing a crucial role in IBS pathophysiology.