PB1985 THE INFLUENCE OF IL1B PROMOTER METHYLATION PROFILE ON FUNCTIONAL STATE OF HEMATOPOIETIC MICROENVIRONMENT

Background: Interleukin‐1b (IL‐1b) is not only a key factor of inflammation, but also a participant in regulation of hematopoietic stem cells (HSCs) and stromal microenvironment. It is known that IL‐1b affect self‐renewal of HSCs and drives their differentiation towards myeloid progenitors. At the same time, IL‐1b promotes expansion of hematopoietic territory by stimulating proliferation and differentiation of stromal precursor cells. This can be achieved upon irradiation, which induces IL‐1b expression in bones. The elevated level of IL‐1b persists a prolonged time until death of irradiated animal. One of possible explanations of this phenomenon could be demethylation of CpG dinucleotides in Il1b promoter. Aims: The aim of this study was to determine if ionizing radiation can trigger alterations in Il1b promoter and result in stable changes in its expression as a consequence. Methods: CBF1 mice (C57Bl/6J x CBA) were irradiated at a dose of 6 Gy with cobalt‐60 source. After 1, 7, 60, 270 and 600 days after irradiation, DNA was isolated from the bones of irradiated and control mice. Bisulfite conversion of DNA was performed followed by PCR. The PCR product was cloned into a plasmid and competent E. coli cells were transformed. Individual colonies of transformants with the insert were selected by blue‐white test. DNA was isolated from 15‐25 white colonies and the insert sequence was determined by Sanger sequencing. The extent of CpG methylation in the initial cell population was determined by the ratio of colonies with the C or T signal in the positions corresponding to CpG. The extent of CpG methylation in the Il1b promoter was also determined in the total cell population by direct sequencing of the converted PCR product. Results: It was shown that, in general, the CpG methylation profile in the Il1b promoter does not change after irradiation. Both in the control and in the experimental group, CpGs in the proximal (‐154 – ‐379 bp from the start of transcription) and in the distant (‐3324 – ‐3629 bp) regions were most methylated (the ratio of methylated CpGs = 0.7‐0.9). On the contrary, CpG, located in the middle (‐2152 – ‐2490 bp), were the least methylated (the ratio of methylated < 0.6). A statistically significant decrease in methylation of individual cytosines was detected after 60 days (‐317 bp) and 600 days (‐194 bp) after irradiation, however, these changes were unstable and were not observed for other periods after irradiation. The functional significance of these changes remains to be established. This study demonstrated that ionizing radiation can alter the methylation of individual CpGs in gene promoters. Summary/Conclusion: It is shown that irradiation can lead to alterations in the methylation profile of the Il1b promoter in bone cells, which can exert influence on the functioning and maintenance of HSCs within osteoblastic niches. This may affect the quality of hematopoiesis and the regulation of inflammatory processes. Probably, hypomethylation agents used to treat patients with hematological malignancies affect not only tumor cells, but also cells of the hematopoietic microenvironment.