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ECPB 2017, 77(1): 23–33
Research articles

Transcriptional Induction of Regulatory Genes Madcam1, S1pr1, Cxcr4 and Ccr7 that Control Lymphocyte Recirculation and Homing in Terms of Experimental Gestational Diabetes Affects T-cells Distribution in Mesenteric Lymph Nodes in Offspring


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 (GD) 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 to 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) is 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 MLN is regulated with adressin Madcam1, chemokine receptors Cxcr4 and Ccr7. And sphingosine-1-phosphate receptors S1pr1 activate T-cell exit from MLN. We studied descendants of intact Wistar rats (males), offspring of rats with experimental gestational diabetes (EGD) and descendants of rats with EGD 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 studied in age 1 and 6 months.

We use RT-PCR method 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 was used glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. 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 the subsets of immunopositive cells in the investigated areas of MLN.

Expression analysis of homing receptors in MLN revealed an expected significant increasing of Ccr7 and Madcam1 mRNA in offsprings of animals with EGD, 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 EGD. Increased expression level of S1PR1 mRNA of MLN 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 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 EGD, 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.

Added 28.02.2017

Keywords: experimental gestational diabetes, mesenteric lymph nodes, Madcam1, Cxcr4, Ccr7, S1pr1

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  1. 1. Aboumrad E, Madec A, Thivolet C. The CXCR4/CXCL12 (SDF-1) signalling pathway protects non-obese diabetic mouse from autoimmune diabetes. Clin Exp Immunol. 2007;148:432-9.
  2. 2. Chew W, Wang W, Herr D. To fingolimod and beyond: The rich pipeline of drug candidates that target S1P signaling. 2016;113:521-532.
  3. 3. Clahsen T, Pabst O, Tenbrock K, Schippers A, Wagner N. Localization of dendritic cells in the gut epithelium requires MAdCAM-1. Clin Immunol. 2015;156(1):74-84.
  4. 4. Comerford I, Harata-Lee Y, Bunting M, Gregor C, Kara E, McColl S. A myriad of functions and complex regulation of the CCR7/CCL19/CCL21 chemokine axis in the adaptive immune system. Cytokine Growth Factor Rev. 2013; 24(3):269-83.
  5. 5. Davalos-Misslitz A, Rieckenberg J, Willenzon S, Worbs T, Kremmer E, Bernhardt G, Förster R. Generalized multi-organ autoimmunity in CCR7-deficient mice. Eur. J. Immunol. 2007;37:613-622.
  6. 6. Davalos-Misslitz A, Rieckenberg J, Willenzon S, Worbs T, Kremmer E, Bernhardt G et al. Generalized multi-organ autoimmunity in CCR7-deficient mice. European Journal of Immunology 2007;37:613-22.
  7. 7. Förster R, Davalos-Misslitz A, Rot A. CCR7 and its ligands: balancing immunity and tolerance. Nat Rev Immunol. 2008;8(5):362-71.
  8. 8. Graham K, Krishnamurthy B, Fynch S, Mollah Z, Slattery R, Santamaria P et al. Autoreactive cytotoxic T lymphocytes acquire higher expression of cytotoxic effector markers in the islets of NOD mice after priming in pancreatic lymph nodes. Am J Pathol. 2011;178(6):2716-25.
  9. 9. Griffith J, Sokol C, Luster A. Chemokines and chemokine receptors: positioning cells for host defense and immunity. Annu Rev Immunol. 2014;32:659-702.
  10. 10. Kawabe T, Sun S, Fujita T, Yamaki S, Asao A, Takahashi T et al. Homeostatic proliferation of naive CD4+ T cells in mesenteric lymph nodes generates gut-tropic Th17 cells. J Immunol. 2013;190(11):5788-98.
  11. 11. Leng Q, Nie Y, Zou Y, Chen J. Elevated CXCL12 expression in the bone marrow of NOD mice is associated with altered T cell and stem cell trafficking and diabetes development. BMC Immunol. 2008;9:51.
  12. 12. Mackley E, Houston S, Marriott C, Halford E, Lucas B, Cerovic V et al. Withers DR CCR7-dependent trafficking of RORγ ILCs creates a unique microenvironment within mucosal draining lymph nodes. Nat Commun. 2015;6:5862.
  13. 13. Macpherson A. Mesenteric lymph nodes at the center of immune anatomy. J. Exp. Med. 2006;203(3):497-500.
  14. 14. Menning A, Hopken U, Siegmund K, Lipp M, Hamann A, Huehn J. Distinctive role of CCR7 in migration and functional activity of naive- and effector/memory-like Treg subsets. European Journal of Immunology 2007;37:1575-83.
  15. 15. Moschovakis G, Förster R. Multifaceted activities of CCR7 regulate T-cell homeostasis in health and disease. Eur J Immunol. 2012;42(8):1949-55.
  16. 16. Olmos S, Stukes S, Ernst J. Ectopic activation of Mycobacterium tuberculosis specific CD4+ T cells in lungs of CCR7-/- mice. Journal of Immunology 2010;184:895-901.
  17. 17. Pabst O, Wahl B, Bernhardt G, Hammerschmidt S. Mesenteric lymph node stroma cells in the generation of intestinal immune responses. J Mol Med (Berl). 2009;87(10):945-51.
  18. 18. Penaranda C, Tang Q, Ruddle N, Bluestone J. Prevention of diabetes by FTY720-mediated stabilization of peri-islet tertiary lymphoid organs. Diabetes. 2010;59(6):1461-8.
  19. 19. Phillips J, Haskins K, Cooke A. MAdCAM-1 is needed for diabetes development mediated by the T cell clone, BDC-2.5. Immunology. 2005;116(4):525-31.
  20. 20. Rivera J, Proia R, Olivera A. The alliance of sphingosine-1-phosphate and its receptors in immunity. Nat Rev Immunol. 2008;8(10):753-63.
  21. 21. Schulz O, Hammerschmidt S, Moschovakis G, Förster R. Chemokines and Chemokine Receptors in Lymphoid Tissue Dynamics. Annu Rev Immunol. 2016;34:203-42.
  22. 22. Shan Z, Xu B, Mikulowska-Mennis A, Michie S. CCR7 directs the recruitment of T cells into inflamed pancreatic islets of nonobese diabetic (NOD) mice. Immunol Res. 2014;58(2-3):351-7.
  23. 23. Spiegel S, Milstien S. The outs and the ins of sphingosine-1-phosphate in immunity. Nat Rev Immunol. 2011;11(6):403-415.
  24. 24. Tarabar D, Hirsch A, Rubin D. Vedolizumab in the treatment of Crohn's disease. Expert Rev Gastroenterol Hepatol. 2016;10(3):283-90.
  25. 25. Tsuji T, Inoue M, Yoshida Y, Fujita T, Kaino Y, Kohno T. Therapeutic approach for type 1 diabetes mellitus using a novel immunomodulator, FTY720 (Fingolimod), in combination with once daily injection of insulin glargine, examined in non-obese diabetic (NOD) mice. J Diabetes Invest 2012;3:132-137.
  26. 26. Vidaković M, Grdović N, Dinić S, Mihailović M, Uskoković A, Jovanović J. The Importance of the CXCL12/CXCR4 Axis in Therapeutic Approaches to Diabetes Mellitus Attenuation. Front Immunol. 2015;
  27. 27. Worbs T, Bode U, Yan S, Hoffmann M, Hintzen G, Bernhardt G, Förster R, Pabst O. Oral tolerance originates in the intestinal immune system and relies on antigen carriage by dendritic cells. J. Exp. Med. 2006;203:519-527.
  28. 28. Xu B, Cook R, Michie S. Alpha4beta7 integrin/MAdCAM-1 adhesion pathway is crucial for B cell migration into pancreatic lymph nodes in nonobese diabetic mice. J Autoimmun. 2010;35(2):124-9.
  29. 29. Yano T, Liu Z, Donovan J, Thomas M, Habener J. Stromal cell derived factor-1 (SDF-1)/CXCL12 attenuates diabetes in mice and promotes pancreatic beta-cell survival by activation of the prosurvival kinase Akt. Diabetes. 2007;56:2946-57.

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