Changes in the Transcriptional Activity of the Lymphocyte Homing Regulatory Genes Madcam1, Cxcr3, Ccr7 and S1pr1 Affect Structure of the Population of T-bet+, Rorγt+ and Foxp3+ cells in Mesenteric Lymph Nodes

Background and aims: Formation of peripheral immunological tolerance (PIT) to self-antigens is an important mechanism for preventing the development of autoimmune diseases. Maternal hyperglycemia that develops in gestational diabetes can influence on the morphogenesis of the immune system and leads to violations of PIT formation to pancreatic antigens. Using mucosa is an attractive way for treatment by administering antigens as tolerogen, especially in children. In animal models oral or intranasal administration of antigen can induce PIT. Mesenteric lymph nodes (MLN) are a major transition point for recirculating lymphocytes of gastrointestinal associated lymphoid tissue and at the same time - the main places for PIT induction. Homing of lymphocytes in mesenteric lymph nodes is regulated with adressin MAdCAM-1, chemokine receptors CXCR4 and CCR7, while sphingosine-1-phosphate receptors S1PR1 activate T-cell exit from mesenteric lymph nodes. The aim of current research was to evaluate the levels of mRNA expression of MAdCAM-1, S1PR1, CXCR4, and CCR7 genes and their effect on the distribution of Th1-, Th17-, and Treg-cells in mesenteric lymph nodes in the offspring of rats with experimental gestational diabetes (EGD) and under conditions of oral insulin administration.  Material and methods: We studied descendants of intact Wistar rats (males), offspring of rats with experimental gestational diabetes and descendants of rats with experimental gestational diabetes, which received short-acting human insulin orally using a pipette for the first 14 days of life at a dose of 30 IU. Each group was evaluated at 1 and 6 months old age. RT-PCR method was used for investigating of mRNA expression levels of genes Madcam1, Cxcr4, Ccr7 and S1pr1 in MLN of experimental rats. As reference gene to determine the relative value of changes in the expression level of target genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used. Normalized relative quantity of cDNA target genes was determined by the method ΔΔCt. The structure of the population of T-bet+, Rorγt+, Foxp3+-cells (i.e., Th1, Th17 and Treg-lymphocytes) were studied based on the analysis of serial histological sections of MLN and data of their morphometric and densitometric characteristics. We determined the absolute (number of cells per 1 mm2 of area) and relative (%) density of the subsets of immunopositive cells in the investigated areas of MLN. Results: Expression analysis of homing receptors in mesenteric lymph nodes revealed an expected significant increase of CCR7 and MAdCAM-1 mRNA in offspring of animals with experimental gestational diabetes, indicating the activation of the immune cells of the lymphoid tissue of the intestine, which is accompanied by intensification of lymphocytes homing and confirms the involvement of these receptors in the pathogenesis of diabetes mellitus. We were unable to detect changes in the mRNA levels of another regulator - CXCR4 in MLN of the offspring of rats with experimental gestational diabetes. Increased expression level of S1PR1 mRNA in MLN nodes lymphocytes in the offspring of animals with diabetes confirms its important role in the progression of diabetes. Signals of chemokine receptors affect the activation of different Th cells subsets and we may assume their pivotal role in the development of autoimmune diseases, particularly diabetes type 1, through violation of oral tolerance. Oral tolerance is generated exclusively in MLN through antigens that are transported from the intestinal surface by DCs through the afferent lymphatics. Thus it is possible to establish communication between the homing regulators and various subsets of Th cells. We recorded a significant increase of T-bet+ and Rorγt+-cells in the offspring of rats with experimental gestational diabetes, which corresponds to the activation of pro-inflammatory lymphocytes Th1 and Th17. At the same time, we observed a decrease of Foxp3+-cells, Treg-lymphocytes responsible for the suppression of the immune response. Conclusions: The changes we detected in the level of expression of the regulators of lymphocyte "homing" in MLN affect the distribution of Th1-, Th17- and Treg-cells in the offspring of rats which parents had EGD. The development of oral insulin tolerance is accompanied by a decrease in the relative normalized mRNA number of Ccr7, Madcam1 and S1pr1 genes in MLN cells, total Rorγt + - and T-bet + lymphocyte density.