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

Abstract

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.