The study examined (a) whether there is sex difference in spinal cord and plasma oxidative stress profiles in Dark Agouti rats immunised for experimental autoimmune encephalomyelitis (EAE), the principal experimental model of multiple sclerosis, and (b) whether there is correlation between the oxidative stress in spinal cord and neurological deficit. Regardless of rat sex, with the disease development xanthine oxidase (XO) activity and inducible nitric oxide synthase (iNOS) mRNA expression increased in spinal cord, whereas glutathione levels decreased. This was accompanied by the rise in spinal cord malondialdehyde level. On the other hand, with EAE development superoxide dismutase (SOD) activity decreased, while O concentration increased only in spinal cord of male rats. Consequently, SOD activity was lower, whereas O concentration was higher in spinal cord of male rats with clinically manifested EAE. XO activity and iNOS mRNA expression were also elevated in their spinal cord. Consistently, in the effector phase of EAE the concentration of advanced oxidation protein product (AOPP) was higher in spinal cord of male rats, which exhibit more severe neurological deficit than their female counterparts. In as much as data obtained in the experimental models could be translated to humans, the findings may be relevant for designing sex-specific antioxidant therapeutic strategies. Furthermore, the study indicated that the increased pro-oxidant-antioxidant balance in plasma may be an early indicator of EAE development. Moreover, it showed that plasma AOPP level may indicate not only actual activity of the disease, but also serve to predict severity of its course.
Given that granulocyte macrophage colony-stimulating factor (GM-CSF) is identified as the key factor to endow auto-reactive Th cells with the potential to induce neuroinflammation in experimental autoimmune encephalomyelitis (EAE) models, the frequency and phenotype of GM-CSF-producing (GM-CSF+) Th cells in draining lymph nodes (dLNs) and spinal cord (SC) of Albino Oxford (AO) and Dark Agouti (DA) rats immunized for EAE were examined. The generation of neuroantigen-specific GM-CSF+ Th lymphocytes was impaired in dLNs of AO rats (relatively resistant to EAE induction) compared with their DA counterparts (susceptible to EAE) reflecting impaired CD4+ lymphocyte proliferation and less supportive of GM-CSF+ Th cell differentiation dLN cytokine microenvironment. Immunophenotyping of GM-CSF+ Th cells showed their phenotypic heterogeneity in both strains and revealed lower frequency of IL-17+IFN-γ+, IL-17+IFN-γ-, and IL-17-IFN-γ+ cells accompanied by higher frequency of IL-17-IFN-γ- cells among them in AO than in DA rats. Compared with DA, in AO rats was also found (i) slightly lower surface density of CCR2 (drives accumulation of highly pathogenic GM-CSF+IFN-γ+ Th17 cells in SC) on GM-CSF+IFN-γ+ Th17 lymphocytes from dLNs, and (ii) diminished CCL2 mRNA expression in SC tissue, suggesting their impaired migration into the SC. Moreover, dLN and SC cytokine environments in AO rats were shown to be less supportive of GM-CSF+IFN-γ+ Th17 cell differentiation (judging by lower expression of mRNAs for IL-1β, IL-6 and IL-23/p19). In accordance with the (i) lower frequency of GM-CSF+ Th cells in dLNs and SC of AO rats and their lower GM-CSF production, and (ii) impaired CCL2 expression in the SC tissue, the proportion of proinflammatory monocytes among peripheral blood cells and their progeny (CD45hi cells) among the SC CD11b+ cells were reduced in AO compared with DA rats. Collectively, the results indicate that the strain specificities in efficacy of several mechanisms controlling (auto)reactive CD4+ lymphocyte expansion/differentiation into the cells with pathogenic phenotype and migration of the latter to the SC contribute to AO rat resistance to EAE.
An accumulating body of evidence suggests that development of autoimmune pathologies leads to thymic dysfunction and changes in peripheral T-cell compartment, which, in turn, perpetuate their pathogenesis. To test this hypothesis, thymocyte differentiation/maturation in rats susceptible (Dark Agouti, DA) and relatively resistant (Albino Oxford, AO) to experimental autoimmune encephalomyelitis (EAE) induction was examined. Irrespective of strain, immunization for EAE (i) increased the circulating levels of IL-6, a cytokine causally linked with thymic atrophy, and (ii) led to thymic atrophy reflecting partly enhanced thymocyte apoptosis associated with downregulated thymic IL-7 expression. Additionally, immunization diminished the expression of Thy-1, a negative regulator of TCRαβ-mediated signaling and activation thresholds, on CD4+CD8+ TCRαβlo/hi thymocytes undergoing selection and thereby impaired thymocyte selection/survival. This diminished the generation of mature CD4+ and CD8+ single positive TCRαβhi thymocytes and, consequently, CD4+ and CD8+ recent thymic emigrants. In immunized rats, thymic differentiation of natural regulatory CD4+Foxp3+CD25+ T cells (nTregs) was particularly affected reflecting a diminished expression of IL-7, IL-2 and IL-15. The decline in the overall thymic T-cell output and nTreg generation was more pronounced in DA than AO rats. Additionally, differently from immunized AO rats, in DA ones the frequency of CD28- cells secreting cytolytic enzymes within peripheral blood CD4+ T lymphocytes increased, as a consequence of thymic atrophy-related replicative stress (mirrored in CD4+ cell memory pool expansion and p16INK4a accumulation). The higher circulating level of TNF-α in DA compared with AO rats could also contribute to this difference. Consistently, higher frequency of cytolytic CD4+ granzyme B+ cells (associated with greater tissue damage) was found in spinal cord of immunized DA rats compared with their AO counterparts. In conclusion, the study indicated that strain differences in immunization-induced changes in thymopoiesis and peripheral CD4+CD28- T-cell generation could contribute to rat strain-specific clinical outcomes of immunization for EAE.
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