Alexander D. Lukin

Vitamin D deficiency is associated with the risk of atopic diseases and respiratory infections. The activated vitamin D receptor (VDR) forms a dimer with the retinoid X receptor alpha (RXRA) and binds to VDR/RXRA composite elements (CEs) in enhancers of target genes. However, VDR/RXRA CEs are identified in only 11.5% of cases in ChIP-Seq peaks. Our hypothesis was that VDR could form a VDR-Partner complex with transcription factor for which CEs have not yet been identified. We utilized Web-MCOT to search for novel VDR/Partner CEs in regulatory DNA. The potential formation of the VDR-Partner protein complex was assessed using the AlphaFold machine learning model. Through real-time RT-PCR, we measured the expression of immune system genes in a culture of U937 macrophage-like cells incubated with the active metabolite of vitamin D, calcitriol. We have predicted novel VDR/NR2C2 and VDR/PPARG CEs in the regulatory regions of immune system genes. We found potential synergism of VDR/NR2C2 and VDR/RXRA CEs in relation to the IRF5 gene, as well as potential synergism of VDR/PPARG and VDR/RXRA CEs for MAPK13. Predicting new regulatory relationships through the identification of new potential VDR/Partner CEs may provide insight into the deep mechanisms of vitamin D involvement in the pathogenesis of atopic dermatitis, bronchial asthma, allergic rhinitis, and pulmonary infections.

Background: Invasion of microorganisms into the gut of insects triggers a cascade of immune reactions accompanied by increased synthesis of effectors (such as antimicrobial peptides, cytokines, and amino acids), leading to changes in the physiological state of the host. We hypothesized that even an inactivated bacterium can induce an immune response in an insect. The aim of this study was to compare the roles of reactive oxygen species (ROS) formation and of the response of detoxification and antioxidant systems in a Colorado potato beetle (CPB) larval model in the first hours after invasion by either an inactivated or live bacterium. Methods: The influence of per os inoculation with inactivated entomopathogenic bacterium Bacillus thuringiensis var. tenebrionis (Bt) on the survival and physiological and biochemical parameters of CPB larvae was assessed as changes in the total hemocyte count (THC), activity of phenoloxidases (POs), glutathione-S-transferases (GSTs), nonspecific esterases (ESTs), catalase, peroxidases, superoxide dismutases (SODs) and formation of reactive oxygen species (ROS). Results: A series of changes occurred within the hemolymph and the midgut of CPBs inoculated with inactivated Bt at 12 h after inoculation. These physiological and biochemical alterations serve to mediate generalized resistance to pathogens. The changes were associated with an increase in the THC and a 1.4–2.2-fold enhancement of detoxification enzymatic activities (such as GST and EST) as well as increased levels of antioxidants (especially peroxidases) in hemolymph in comparison to the control group. Suppressed EST activity and reduced ROS formation were simultaneously detectable in the larval midgut. Inoculation of beetle larvae with active Bt cells yielded similar results (elevated THC and suppressed PO activity). A fundamental difference in the immune activation processes between larvae that ingested the inactivated bacterium and larvae that had consumed the active bacterium was that the inactivated bacterium did not influence ROS formation in the hemolymph but did reduce their formation in the midgut. At 24 h postinfection with active Bt, ROS levels went up in both the hemolymph and the midgut. This was accompanied by a significant 5.7-fold enhancement of SOD activity and a 5.3-fold suppression of peroxidase activity. The observed alterations may be due to within-gut toxicity caused by early-stage bacteriosis. The imbalance in the antioxidant system and the accumulation of products toxic to the “putative” pathogen can activate detoxification mechanisms, including those of an enzymatic nature (EST and GST). The activation of detoxification processes and of innate immune responses is probably due to the recognition of the “putative” pathogen by gut epithelial cells and is similar in many respects to the immune response at early stages of bacteriosis.