ANALYSIS OF CYTOARCHITECTONICS OF TLR2⁺ AND TLR4⁺ LYMPHOCYTES AND TRANSCRIPTIONAL ACTIVITY OF THE GENES Gp2, Spi-B, Nf-kB1, с-REL, TNFα AND TNFr IN GALT OF RATS IN EXPERIMENTAL DIABETES MELLITUS AND AFTER PENTOXIFYLLINE ADMINISTRATION

Summary.

Changes in the state of gut-associated lymphoid tissue (GALT) and the composition of the intestinal microbiome, both in experimental STZ-induced diabetes and in development of type 1 diabetes in humans as well as chronic inflammation due to stimulation of innate immunity are crucially important in the development of type 1 diabetes mellitus. One of the most important mediators for interactions between the intestinal microbiome and GALT are specialized M cells of the follicle-associated epithelium, providing transcytotic delivery of antigens to the underlying lymphoid structures. TNFα-signaling also plays a supporting role in the formation of M cells. Therefore, the aim of our work was to study some features of TLRs expression and transcriptional activity of the Gp2, Spi-B, Nf-kB1, c-Rel, TNFα and TNFr genes in GALT in experimental diabetes mellitus (EDM), and after pentoxifylline administration. To identify TLR2⁺ cells and TLR4⁺ cells, an immunofluorescence method was used with monoclonal antibodies to corresponding pattern-recognizing receptors. To study the transcriptional activity of genes, the method of real-time reverse transcription polymerase chain reaction (RT-PCR) was used. In the course of developing experimental pathology, at the terms of 2 and 4 weeks, a decrease in the total density of TLR2⁺ and TLR4⁺ lymphocytes was observed in lamina propria of villus (villus) and subepithelial zone isolated lymphoid follicles (ILF) of rat ileum. At the same time, the density of TLR2 on the membrane of immunopositive cells was increased for small lymphocytes, and TLR4 density has became higher in medium and small lymphocytes. The pentoxifylline administration to diabetic rats resulted in a decrease in the total density of TLR2⁺ cells at the 2nd week of development of the pathology, and an increase in this index at the 4th week. The total density of TLR4⁺ cells showed changing growth rates only in villus at the 2nd week of EDM development in the presence of pentoxifylline. Changes in the density of TLR2 and TLR4 on the surface of lymphocytes were multidirectional. The development of diabetes is also reflected in the transcriptional induction of genes of the key transcription factors NF-kB1 and c-Rel in GALT cells at both the 2nd and 4th week of the development of EDM. Meanwhile, administration of pentoxifylline resulted in a significantly reduced level of normalized expression of NF-kB1 mRNA during the entire observation period and increased this indicator for c-Rel mRNA at the 2nd week. The growth of normalized expression of markers of M cells Gp2 and Spi-B was observed both on the 2nd and on the 4th week of the development of experimental pathology. Administration of pentoxifylline to diabetic animals was largely reflected in the change in the intensity of mRNA expression of the mature M cell Gp2 marker. This parameter was increased during the 2nd week of developing pathology, and on the 4th week, a downward trend was shown. The development of EDM led to a significantly increased level of near-normalized expression of proinflammatory TNFα cytokine and its receptor TNFr, and demonstrated a trend towards their decrease following pentoxifylline administration in diabetic animals.

1. Degen A.S., Kamyshnyi A.M. Expression of cytoplasmic nod-2 and rig-i receptors of innate immunity in intestine of rats in experimental diabetes mellitus. Rossiyskiy immunologicheskiy zhurnal = Russian Journal of Immunology, 2014, no. 3, pp. 525-528. (In Russ.)

2. Alyanakian M.A., Grela F., Aumeunier A., Chiavaroli C., Gouarin C., Bardel E., Normier G., Chatenoud L., Thieblemont N., Bach J.F. Transforming growth factor-beta and natural killer T-cells are involved in the protective effect of a bacterial extract on type 1 diabetes. Diabetes, 2006, Vol. 55, no. 1, pp. 179-185.

3. Biswas A., Banerjee P., Biswas T. Porin of Shigella dysenteriae directly promotes toll-like receptor 2-mediated CD4 + T cell survival and effector function. Mol. Immunol., 2009, Vol. 46, no. 15, pp. 3076-3085.

4. d’Addio F., Fiorina P. Type 1 diabetes and dysfunctional intestinal homeostasis. Trends Endocrinol. Metab., 2016, Vol. 27, no. 7, pp. 493-503.

5. Degen A., Kamyshny A. Distribution characteristics of RIG-I receptors of innate immunity in experimental diabetes mellitus and administration of nonspecific blockers of TNF-α. J. Immunol. Clin. Microbiol., 2018, Vol. 3, no. 3, pp. 50-59.

6. Devaraj S., Dasu M.R., Rockwood J., Winter W., Griffen S.C., Jialal I. Increased toll-like receptor (TLR) 2 and TLR4 expression in monocytes from patients with type 1 diabetes: further evidence of a proinflammatory state. J. Clin. Endocrinol. Metab., 2008, no. 93, pp. 578-583.

7. Dong C. TH17 cells in development: an updated view of their molecular identity and genetic programming. Nat. Rev. Immunol., 2008, no. 8, pp. 337-348.

8. Faustman D.L. TNF, TNF inducers, and TNFR2 agonists: A new path to type 1 diabetes treatment. Diabetes Metab. Res. Rev., 2018, Vol. 34, no. 1.

9. Flaherty S., Reynolds J.M. TLR function in murine CD4(+) T lymphocytes and their role in inflammation. Methods Mol Biol., 2016, no. 1390, pp. 215-227.

10. Fulford T.S., Ellis D., Gerondakis S. Understanding the roles of the NF-κB pathway in regulatory T cell development, differentiation and function. Prog. Mol. Biol. Transl. Sci., 2015, no. 136, pp. 57-67.

11. Hase K., Kawano K., Nochi T., Pontes G.S., Fukuda S., Ebisawa M., Kadokura K., Tobe T., Fujimura Y., Kawano S., Yabashi A., Waguri S., Nakato G., Kimura S., Murakami T., Iimura M., Hamura K., Fukuoka S., Lowe A.W., Itoh K., Kiyono H., Ohno H. Uptake through glycoprotein 2 of FimH1 bacteria by M cells initiates mucosal immune response. Nature, 2009, Vol. 462, no. 7270, pp. 226-230.

12. Imanishi T., Hara H., Suzuki S., Suzuki N., Akira S., Saito T. Cutting edge: TLR2 directly triggers Th1 effector functions. J. Immunol., 2007, no. 178, pp. 6715-6719.

13. Jialal I., Yun J.M., Bremer A., Devaraj S. Demonstration of increased TLR2 and TLR4 Expression in monocytes of type 1 diabetic patients with microvascular complications. Metabolism, 2011, Vol. 60, no. 2, pp. 256-259.

14. Kanaya T., Hase K., Takahashi D., Fukuda S., Hoshino K., Sasaki I., Hemmi H., Knoop K.A., Kumar N., Sato M., Katsuno T., Yokosuka O., Toyooka K., Nakai K., Sakamoto A., Kitahara Y., Jinnohara T., McSorley S.J., Kaisho T., Williams I.R., Ohno H. The Ets transcription factor Spi-B is essential for the differentiation of intestinal microfold cells. Nat. Immunol., 2012, Vol. 13, no. 8, pp. 729-736.

15. Knoop K.A., Kumar N., Butler B.R., Sakthivel S.K., Taylor R.T., Nochi T., Akiba H., Yagita H., Kiyono H., Williams I.R. RANKL is necessary and sufficient to initiate development of antigen-sampling M cells in the intestinal epithelium. J. Immunol., 2009, Vol. 183, no. 9, pp. 5738-5747.

16. Koulmanda M., Bhasin M., Awdeh Z., Qipo A., Fan Z., Hanidziar D., Putheti P., Shi H., Csizuadia E., Libermann T.A., Strom T.B. The role of TNF-α in mice with type 1and 2diabetes. PLoS ONE, 2012, Vol. 7, no. 5, pp. 332-354.

17. Liang L., Beshay E., Prud’homme G.J. The phosphodiesterase inhibitors pentoxifylline and rolipram prevent diabetes in NOD mice. Diabetes, 1998, Vol. 47, no. 4, pp. 570-575.

18. Maloy K.J., Powrie F. Intestinal homeostasis and its breakdown in inflammatory bowel disease. Nature, 2011, no. 474, pp. 298-306.

19. Nakamura Y., Kimura S., Hase K. M cell-dependent antigen uptake on follicle-associated epithelium for mucosal immune surveillance. Inflamm. Regen., 2018, no. 38, pp. 1-5.

20. Needell J.C., Zipris D. The Role of the intestinal microbiome in type 1 diabetes pathogenesis. Curr. Diab. Rep., 2016, Vol. 16, no. 10, pp. 8-9.

21. Ohno H. Intestinal M cells. J. Biochem., 2016, Vol. 159, no. 2, pp. 151-160.

22. Opazo M.C., Ortega-Rocha E.M., Coronado-Arrázola I., Bonifaz L.C., Boudin H., Neunlist M., Bueno S.M., Kalergis A.M., Riedel C.A.. Intestinal microbiota influences non-intestinal related autoimmune diseases. Front. Microbiol., 2018, Vol. 9, 432. doi: 10.3389/fmicb.2018.00432.

23. Qiao Y.C., Chen Y.L., Pan Y.H., Tian F., Xu Y., Zhang X.X., Zhao H.L. The change of serum tumor necrosis factor alpha in patients with type 1 diabetes mellitus: A systematic review and meta-analysis. PLoS ONE, 2017, Vol. 12, no. 4, e0176157. doi: 10.1371/journal.pone.0176157.

24. Ramakrishnan P., Yui M.A., Tomalka J.A., Majumdar D., Parameswaran R., Baltimore D. Deficiency of nuclear factor-κB c-Rel accelerates the development of autoimmune diabetes in NOD mice. Diabetes, 2016, Vol. 65, no. 8, pp. 2367-2379.

25. Sato S., Kaneto S., Shibata N., Takahashi Y., Okura H., Yuki Y., Kunisawa J., Kiyono H. Transcription factor Spi-B-dependent and -independent pathways for the development of Peyer’s patch M cells. Mucosal Immunol., 2013, Vol. 6, no. 4, pp. 838-846.

26. Taylor R.T., Patel S.R., Lin E., Butler B.R., Lake J.G., Newberry R.D., Williams I.R. Lymphotoxin-independent expression of TNF-related activation-induced cytokine by stromal cells in cryptopatches, isolated lymphoid follicles, and Peyer’s patches. J. Immunol., 2007, Vol. 178, no. 9, pp. 5659-5667.

27. Vaarala O. Human intestinal microbiota and type 1 diabetes. Curr. Diab. Rep., 2013, Vol. 13, no. 5, pp. 601-607.

28. Visser J., Groen H., Klatter F., Rozing J. Timing of pentoxifylline treatment determines its protective effect on diabetes development in the Bio Breeding rat. Eur. J. Pharmacol., 2002, Vol. 445, no. 1-2, pp. 133-140.

29. Wood M.B., Rios D., Williams I.R.TNF-α augments RANKL-dependent intestinal M cell differentiation in enteroid cultures. Am. J. Physiol. Cell Physiol., 2016, Vol. 311, no. 3, pp. 498-507.

30. Zanin-Zhorov A., Tal-Lapidot G., Cahalon L., Cohen-Sfady M., Pevsner-Fischer M., Lider O., Cohen I.R. Cutting edge: T cells respond to lipopolysaccharide innately via TLR4 signaling. J. Immunol., 2007, Vol. 179, no. 1, pp. 41-44.

31. Zhang H., Bi J., Yi H., Fan T., Ruan Q., Cai L., Chen Y.H., Wan X. Silencing c-Rel in macrophages dampens Th1 and Th17 immune responses and alleviates experimental autoimmune encephalomyelitis in mice. Immunol. Cell Biol., 2017, Vol. 95, no. 7, pp. 593-600.