Annotation of the results obtained in 2023
Multiple sclerosis (MS) is a severe and currently incurable chronic autoimmune disease that mainly affects young people aged 20 - 40 years. In recent years, evidence has been obtained that reactive oxygen species (ROS) play a significant role in the induction and pathogenesis of MS (as well as its model, experimental autoimmune encephalomyelitis, EAE). ROS are actively released by monocytes during any inflammation and induce morphological changes in the cerebral vascular endothelium, and these changes pave the way for lymphocyte infiltration through the blood-brain barrier (BBB). The present study was undertaken to test the hypothesis that the administration of exogenous peroxiredoxin 6 (Prdx6) can help shift the disturbed redox balance and restore the BBB damaged by chronic autoimmune inflammatory processes, or at least slow down its damage. We hypothesized that restoration of the BBB would prevent activated lymphocytes from entering the CNS and could slow or stop the progression of this chronic, self-supporting autoimmune inflammation. In addition, we hypothesized that such therapy may have a synergistic effect with a immunomodulatory therapy aimed at reducing T cell-mediated inflammation. In the present work, in particular, we studied the combination of Prdx6 with fingolimod, which belongs to the most advanced technologies for the treatment of multiple sclerosis at the moment - antilymphocyte drugs, which are modulators of sphingosine-1-phosphate (S1P) receptors. These drugs prevent the release of lymphocytes from peripheral lymphoid organs. The non-selective S1P receptor modulator fingolimod has been clinically tested and approved in the USA and Russia for the treatment of relapsing-remitting MS. However, S1P has a pleiotropic effect, and the integrity of the BBB depends on S1P signaling, so S1P blockers may impair vascular barrier function. Currently, intensive work is performed to test selective modulators of different groups of S1P receptors or to search for drugs aimed, beside to S1P receptors, to other targets, however, it should be noted that the beneficial effect of S1P on lymphocytes and the undesirable effect of S1P on the BBB can be realized through the same S1P1 receptor. There is reason to believe that improving the state of the BBB by administering Prdx6 to the patient will increase the effectiveness of S1P modulators. The administration of fingolimod as monotherapy led to an improvement in the general health status of the animals. Symptoms that developed were less severe than those in the untreated group, but after discontinuation of fingolimod, symptoms began to worsen and approached those of the untreated group at the end of the follow-up period. Animals that received Prdx6 before the administration of fingolimod showed attenuated symptoms similar to those in the fingolimod group during the initial period of the disease, but after discontinuation of fingolimod their symptoms not only did not worsen, but even improved up to almost complete recovery. Thus, the effect of combining Prdx6 with fingolimod was apparently beneficial. Prdx6 produced a stimulating effect on cells of the immune system according to analysis of signaling cascades. Moreover, in the context of autoimmune inflammation, it increased and activated proapoptotic and antiproliferative cascades in lymphocytes, but did not cause hyperactivation, which is favorable. Administration of fingolimod to mice with EAE significantly reduced lymphocyte activation, assessed by NF-kB p65 protein phosphorylation, compared with untreated mice. However, its effect on the pro-apoptotic and anti-proliferative cascades was negligible. On the contrary, co-administration of fingolimod with Prdx6 increased the activation of pro-apoptotic (caspase-3) and anti-proliferative (p53) cascades, which should be considered beneficial in conditions of autoimmune inflammation and proliferation of auto-aggressive lymphocyte clones. Fingolimod monotherapy resulted in a slight decrease in the levels of the proinflammatory cytokines IFN-gamma and IL-17 on the early stage of the disease, but at a later stage, when fingolimod was not administered, the levels of inflammatory cytokines returned to high values. Co-administration of fingolimod and Prdx6 led to a small but non-significant decrease in pro-inflammatory cytokines at an early stage, but at a late stage the level of all cytokines studied was almost normalized. Thus, the introduction of Prdx6 allowed to prolong the effect of fingolimod. Induction of EAE caused a sharp disruption of BBB permeability, assessed by the accumulation of Evan’s blue dye. Prdx6 reduced EAE-induced BBB disruption in earlier but not later stages of EAE. Fingolimod as monotherapy was effective in the early stage of the disease, while at a later stage BBB permeability increased again. The combination of fingolimod+Prdx6 reduced both early and late-stage permeability, although not to a greater extent than Prdx6 alone. Analysis of the state of the BBB by the level of intercellular junction proteins (TJP) showed a significant decrease in the levels of occludin-1, claudin-1 and ZO-1 in brain tissue. The administration of fingolimod to EAE-bearing mice increased the decline in the levels of occludin-1 and claudin-1, which is obviously an unfavorable effect of the drug. The use of Prdx6 in combination with fingolimod reduced the drop in occludin-1 and claudin-1 levels in almost all cases, negating the harmful side effects of fingolimod and improving the health of the BBB. Also, Prdx6, alone and in combination with fingolimod and thymulin, reduced lymphocyte infiltration into the brain. Analysis of the expression of several proteins involved in the oxidative and inflammatory response in the brain showed that the EAE-induced increase in the expression of NADPH oxidase NOX1 was blocked by any of Prdx6 and fingolimod or their combination. NOX4 expression, which was also increased in EAE, was reduced by Prdx6 at the late stage but not at the early stage, whereas fingolimod blocked NOX4 expression at the early stage, but it began to increase later. Co-administration of Prdx6 and fingolimod blocked NOX4 expression at both stages. The relationship of Prdx6 with the expression of NADPH oxidases, which may be one of the mechanisms of the beneficial effects of drugs on the BBB at the molecular level, is an interesting topic for future research. We may conclude that the hypothesis that exogenous Prdx6 could help repair the damaged BBB or slow down its damage was essentially justified. In addition, it was demonstrated that Prdx6 reduced the negative effect of fingolimod on BBB permeability.

 

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Annotation of the results obtained in 2022
During the reporting period, an analysis of the literature was carried out and a review article was written and published in a journal from the Q1 quartile. The article was dedicated to the effects of systemic regulators of the inflammatory response, including thymic hormones such as thymulin, on intracellular signaling cascades associated with the regulation of cell proliferation and senescence, both in normal conditions and in inflammatory diseases. The analysis presented in this article allowed us to postulate a multilevel system of central regulators of inflammatory processes, including neurotransmitters, glucocorticoids, and thymic hormones. According to the proposed model, this system finely regulates the cell cycle and metabolic/catabolism processes depending on the level of systemic stress, the stage of the stress response, and the body's energy capabilities, shifting the balance between the normal cell cycle, arrest of cell division, senescence, and apoptosis. This analysis allows a better understanding of the regulation of inflammatory and stress responses of cells in normal and pathological conditions. The study was performed to test the hypothesis that the violation of the blood-brain barrier (BBB) may play a significant role in the development of EAE, and that peroxiredoxin 6 can compensate for the breakage of the BBB. In addition, at this stage, it was also decided to test the effect of the combination of peroxiredoxin-6 with thymulin on the general health status of the animals, the state of the BBB and the activation of the immune system. Experimental autoimmune encephalomyelitis (EAE) (which is a model of multiple sclerosis) was induced in SJL mice by administering PLP (proteolipid protein) myelin protein in combination with an adjuvant complex according to the scheme earlier proposed for induction of this model: subcutaneous administration of a suspension of PLP in complete Freund's adjuvant, additionally enriched with Mycobacterium tuberculosis bacteria (4 mg/ml versus 2 mg/ml in standard complete Freund's adjuvant) + intraperitoneal administration of pertussis toxin on the day of induction and after 48 hours. EAE was manifested in mice as an impairment of muscle tone and motor functions, up to paralysis. Symptoms were assessed during the entire duration of the experiment (30 days) according to the scale previously described in the literature: 1 point - loss of tail tone, 2 points - hemiparesis, 3 points - paraparesis, 4 points - paraplegia, 5 points - death. Signs were monitored every other day. This scheme, under our conditions, led to the induction of EAE in 58 out of 60 (96.7%) mice that underwent the procedure. Symptoms began to develop, starting from the 8th day, which corresponds to the literature data. 2 mice that did not develop EAE were excluded from the experiment. The most common symptoms in most mice were loss of tail tone and paralysis of the hind legs; although a full range of more severe signs were observed. During the study, only one mouse died from EAE. According to the literature, this model is also characterized by a relapsing-remitting course (with alternating exacerbations and remissions). Under our conditions, remissions were observed in the group of untreated animals, but a complete cure did not occur. Body weight was also monitored and the data show that mice with EAE lose weight, as it can be expected. At this stage of the project, the following experimental groups of animals were used: 1. Control (SJL mice without treatment), 2. Prdx6 (mice treated with peroxiredoxin-6 without EAE induction) 3. EAE (mice with induced EAE, without treatment) 4. EAE-Prdx6 (EAE mice treated with peroxiredoxin-6) 5. EAE thymulin (EAE mice treated with thymulin) 6. EAE-thymulin-Prdx6 (EAE mice treated with both thymulin and peroxiredoxin 6) An additional group of mice received an increased dose of Prdx6 only twice, immediately before and immediately after EAE induction, and did not receive any additional treatment. This experiment was carried out to test the effect of Prdx6 on EAE induction. Data from this additional experiment showed that Prdx6 somewhat slowed down the onset of symptoms (which developed on days 12-13 instead of day 8), but did not prevent the induction of EAE. Preliminary data on the general health status of the animals showed that the administration of the studied drugs (peroxiredoxin 6 and thymulin) led to a significant improvement in the health status of the animals, assessed by the scale of symptoms and body weight. A significant part of the animals in both groups (treated with Prdx6 or thymulin) showed a complete recovery by the end of the experiment (30 days). Whereas, symptoms of EAE, including severe ones (paralysis), remained in untreated animals. The most encouraging results were obtained in the EAE-thymulin-Prdx6 group, where all the animals recovered from the disease. Throughout the study, in order to monitor the state of the blood-brain barrier, blood was taken from the tail vein of mice for analysis for occludin by dot-blotting. Occludin is a protein of intercellular junctions, and its serum level was reported to increase in patients with multiple sclerosis. Dot blotting was performed in triplicate with the recombinant protein as an internal positive control. Preliminary data show that EAE resulted in an increase in the serum occludin level even before the onset of symptoms of the disease. This supports the hypothesis that disruption of the BBB may be important for the induction of EAE (and possibly multiple sclerosis). It was also shown that peroxiredoxin-6, as well as its combination with thymulin, led to a decrease in the level of occludin in the blood serum, confirming our assumption that peroxiredoxin-6 can improve the impaired BBB. Also, to assess the state of the BBB, the method of assessing its permeability with Evans dye was used, for which some mice from each group were injected with the dye into the tail vein. Then, the dye was washed out of circulation by perfusion of saline through the heart, according to the published procedure. After these manipulations, the brain and spinal cord of mice were collected. The amount of dye that penetrated into the brain through the BBB was assessed by spectrophotometry after brain homogenization and protein precipitation using TCA (for a dye not bound to proteins), as well as by fluorescence microscopy (for the total content of the dye). Preliminary results show that after the onset of the symptoms, the permeability of the BBB to the dye increased significantly, and although the barrier somewhat restored in the later stages, it still remained severely damaged. The introduction of a combination of peroxiredoxin 6 and thymulin improved the state of the BBB in the later stages. These experiments were carried out at the time of two planned sacrifices of mice, described below. Mice were sacrificed in two groups, on days 16 and 30 after EAE induction (to test for early and late effects), and the following biological material was collected: blood serum (for ELISA analysis of pro-inflammatory cytokines associated with various lymphocyte subpopulations), brain (for analysis of the expression of pro-inflammatory factors by PCR (iNOS), and Western blotting (VCAM-1, metalloprotease), spinal cord (for analysis of lymphocyte infiltration by immunohistochemistry), spleen (for analysis of the number of leukocytes and activation of signaling cascades in leukocytes by Western blot. General blood test and leukocyte count were also evaluated using a hemoanalyser. Analysis of the total white cell count showed that EAE reduced this indicator, and the administration of peroxiredoxin 6 and/or thymulin had no significant effect on this decrease. Preliminary analysis of cytokine levels showed that EAE led to an increase in the production of interferon gamma, mainly produced by type 1 T-helpers, both at an early and late stage of EAE development. Thymulin somewhat reduced interferon gamma production at the late stage of EAE, and the combination of peroxiredoxin 6 and thymulin significantly reduced its level. A similar picture was observed for IL-17, produced mainly by T-helpers 17, which, according to the literature, play an important role in the development of multiple sclerosis and EAE. We did not observe a significant effect of EAE on the production of TNF and IL-6 cytokines. Other obtained samples and data are still in processing. Thus, data was obtained on the dynamics of the BBB state in the induced model of multiple sclerosis, as well as on the therapeutic activity of the recombinant antioxidant protein peroxiredoxin 6 and the thymus hormone thymulin, and the best result was observed for combination of both drugs.

 

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