Immunological mechanisms underlying inflammatory bowel disease

Ulcerative colitis (UC) and Crohn‘s disease (CD), collectively referred to as inflammatory bowel diseases (IBD), are characterized by aberrant immune responses in the gut, resulting in chronic intestinal inflammation. There is no cure for IBD, and the disease is increasing world-wide. The pathogenic mechanisms that drive disease onset and chronicity are currently unclear but seem to include a complex mixture of heritable pre-disposition and acquired factors. The inflammation during IBD is characterized by a large influx of immune cells from the circulation to the inflamed intestine. This is mediated by the expression of chemokine receptors on the surface of the leukocytes, allowing them to migrate towards gradients of chemokines produced in the intestinal tissues. However, the mechanisms behind chemokine receptor-mediated leukocyte infiltration to the gut are poorly understood. The papers included in this thesis investigate the roles of CD4+ T helper cells and monocytes, focusing on chemokine receptor interactions, in mediating intestinal inflammation during IBD. Whereas the inductive mechanisms of chemokine receptors during colitis have mainly been studied in murine T cells, we set out to investigate blood monocytes in IBD patients. We found that the chemokine receptor CCR9, important for gut-homing in T cells, is expressed on a subset of monocytes that is increased during active IBD. Furthermore, we could show that a large number of chemokine receptors are up-regulated on IBD monocytes compared to healthy controls, as well as being differentially expressed between ulcerative colitis and Crohn’s disease. As UC and CD may be clinically similar, they are often difficult to distinguish. However, as optimal therapy choices for the respective disorders differ, correctly diagnosing IBD is crucial and thus, chemokine receptor profiling on blood monocytes constitutes a potential diagnostic approach. In T cells, murine data suggests that CCR9 interactions are important for small intestinal homing whereas their role during colitis remains unclear. We have investigated CD4+ T helper cells infiltrating the colonic mucosa during inflammation, and found that CCR9 is widely expressed on these cells, indicating importance role during human colitis. Furthermore, CCR9 expression levels were higher on T cells derived from un-affected compared to inflamed specimens, which might suggest that CCR9-positive cells have a regulatory function. In conclusion, we have shown that chemokine receptor interactions are important for colonic immune responses during IBD. Understanding the complexity of the chemokine receptor system is fundamental to successfully targeting leukocyte migration pathways for therapeutic purposes

Increased CD4+ T cell lineage commitment determined by CpG methylation correlates with better prognosis in urinary bladder cancer patients

BACKGROUND: Urinary bladder cancer is a common malignancy worldwide. Environmental factors and chronic inflammation are correlated with the disease risk. Diagnosis is performed by transurethral resection of the bladder, and patients with muscle invasive disease preferably proceed to radical cystectomy, with or without neoadjuvant chemotherapy. The anti-tumour immune responses, known to be initiated in the tumour and draining lymph nodes, may play a major role in future treatment strategies. Thus, increasing the knowledge of tumour-associated immunological processes is important. Activated CD4+ T cells differentiate into four main separate lineages: Th1, Th2, Th17 and Treg, and they are recognized by their effector molecules IFN-γ, IL-13, IL-17A, and the transcription factor Foxp3, respectively. We have previously demonstrated signature CpG sites predictive for lineage commitment of these four major CD4+ T cell lineages. Here, we investigate the lineage commitment specifically in tumour, lymph nodes and blood and relate them to the disease stage and response to neoadjuvant chemotherapy. RESULTS: Blood, tumour and regional lymph nodes were obtained from patients at time of transurethral resection of the bladder and at radical cystectomy. Tumour-infiltrating CD4+ lymphocytes were significantly hypomethylated in all four investigated lineage loci compared to CD4+ lymphocytes in lymph nodes and blood (lymph nodes vs tumour-infiltrating lymphocytes: IFNG -4229 bp p < 0.0001, IL13 -11 bp p < 0.05, IL17A -122 bp p < 0.01 and FOXP3 -77 bp p > 0.05). Examination of individual lymph nodes displayed different methylation signatures, suggesting possible correlation with future survival. More advanced post-cystectomy tumour stages correlated significantly with increased methylation at the IFNG -4229 bp locus. Patients with complete response to neoadjuvant chemotherapy displayed significant hypomethylation in CD4+ T cells for all four investigated loci, most prominently in IFNG p < 0.0001. Neoadjuvant chemotherapy seemed to result in a relocation of Th1-committed CD4+ T cells from blood, presumably to the tumour, indicated by shifts in the methylation patterns, whereas no such shifts were seen for lineages corresponding to IL13, IL17A and FOXP3. CONCLUSION: Increased lineage commitment in CD4+ T cells, as determined by demethylation in predictive CpG sites, is associated with lower post-cystectomy tumour stage, complete response to neoadjuvant chemotherapy and overall better outcome, suggesting epigenetic profiling of CD4+ T cell lineages as a useful readout for clinical staging

Additional file 1: of Increased CD4+ T cell lineage commitment determined by CpG methylation correlates with better prognosis in urinary bladder cancer patients

Table S1. PCR assay-specific sequencing primers (DOCX 14 kb

Additional file 3: of Increased CD4+ T cell lineage commitment determined by CpG methylation correlates with better prognosis in urinary bladder cancer patients

Figure S1. Analysis of Cisplatin effect on healthy donors CD4+ T cells in vitro. CD4+ T cells were isolated from blood of healthy donors (n = 4) and cultured in vitro in the presence of neoadjuvant chemotherapy drug, Cisplatin. Stimulation at day 0 is indicated on x-axis. Sim = αCD3 and αCD28. Cisp 25 μM cisplatin. At day 6, all cultures were treated with αCD3 and αCD28, and cisplatin cultures (grey bars) received 25 μM cisplatin. The cells were harvested at day 12 for analysis. a Whole genome methylation was measured by 5mC ELISA. Corresponding cultures without cisplatin was used for normalization. Friedman test was used for statistical analysis. b Methylation of IFNG locus was measured. Unstimulated cells from Day 0 was used as normalization. (TIF 840 kb