Aging diminishes the resistance of AO rats to EAE: putative role of enhanced generation of GM-CSF Expressing CD4+T cells in aged rats

Background: Aging influences immune response and susceptibility to EAE in a strain specific manner. The study was designed to examine influence of aging on EAE induction in Albino Oxford (AO) rats. Results: Differently from 3-month-old (young) rats, which were resistant to EAE induction, the majority of aged (24-26-month-old) rats developed mild chronic form of EAE. On 16th day post-immunization, when in aged rats the neurological deficit reached plateau, more mononuclear cells, including CD4+ T lymphocytes was retrieved from spinal cord of aged than young rats. The frequencies of IL-17+ and GM-CSF+ cells within spinal cord infiltrating CD4+ lymphocytes were greater in aged rats. To their increased frequency contributed the expansion of GM-CSF + IL-17 + IFN-gamma+ cells, which are highly pathogenic in mice. The expression of the cytokines (IL-1 beta and IL-23/p19) driving GM-CSF + IL-17 + IFN-gamma + cell differentiation in mice was also augmented in aged rat spinal cord mononuclear cells. Additionally, in aged rat spinal cord the expansion of GM-CSF + IL-17-IFN-gamma- CD4+ T lymphocytes was found. Consistently, the expression of mRNAs for IL-3, the cytokine exhibiting the same expression pattern as GM-CSF, and IL-7, the cytokine driving differentiation of GM-CSF + IL-17-IFN-gamma- CD4 + lymphocytes in mice, was upregulated in aged rat spinal cord mononuclear cells, and the tissue, respectively. This was in accordance with the enhanced generation of the brain antigen-specific GM-CSF+ CD4+ lymphocytes in aged rat draining lymph nodes, as suggested by (i) the higher frequency of GM-CSF+ cells (reflecting the expansion of IL-17-IFN-gamma- cells) within their CD4+ lymphocytes and (ii) the upregulated GM-CSF and IL-3 mRNA expression in fresh CD4+ lymphocytes and MBP-stimulated draining lymph node cells and IL-7 mRNA in lymph node tissue from aged rats. In agreement with the upregulated GM-CSF expression in aged rats, strikingly more CD11b + CD45(int) (activated microglia) and CD45(hi) (mainly proinflammatory dendritic cells and macrophages) cells was retrieved from aged than young rat spinal cord. Besides, expression of mRNA for SOCS1, a negative regulator of proinflammatory cytokine expression in innate immunity cells, was downregulated in aged rat spinal cord mononuclear cells. Conclusions: The study revealed that aging may overcome genetic resistance to EAE, and indicated the cellular and molecular mechanisms contributing to this phenomenon in AO rats

GM-CSF-Producing Th Cells in Rats Sensitive and Resistant to Experimental Autoimmune Encephalomyelitis

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-gamma+, IL-17+ IFN-gamma-, and IL-17-IFN-gamma+ cells accompanied by higher frequency of IL-17-IFN-gamma- 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-gamma+ Th17 cells in SC) on GM-CSF+ IFN-gamma+ 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-gamma+ Th17 cell differentiation (judging by lower expression of mRNAs for IL-1 beta, 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 (CD45(hi) 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

Granulocyte-macrophage colony stimulatory factor enhances the pro-inflammatory response of interferon-γ-treated macrophages to pseudomonas aeruginosa infection

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections at compromised epithelial surfaces, such those found in burns, wounds, and in lungs damaged by mechanical ventilation or recurrent infections, particularly in cystic fibrosis (CF) patients. CF patients have been proposed to have a Th2 and Th17-biased immune response suggesting that the lack of Th1 and/or over exuberant Th17 responses could contribute to the establishment of chronic P. aeruginosa infection and deterioration of lung function. Accordingly, we have observed that interferon (IFN)-γ production by peripheral blood mononuclear cells from CF patients positively correlated with lung function, particularly in patients chronically infected with P. aeruginosa. In contrast, IL-17A levels tended to correlate negatively with lung function with this trend becoming significant in patients chronically infected with P. aeruginosa. These results are in agreement with IFN-γ and IL-17A playing protective and detrimental roles, respectively, in CF. In order to explore the protective effect of IFN-γ in CF, the effect of IFN-γ alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), on the ability of human macrophages to control P. aeruginosa growth, resist the cytotoxicity induced by this bacterium or promote inflammation was investigated. Treatment of macrophages with IFN-γ, in the presence and absence of GM-CSF, failed to alter bacterial growth or macrophage survival upon P. aeruginosa infection, but changed the inflammatory potential of macrophages. IFN-γ caused up-regulation of monocyte chemoattractant protein-1 (MCP-1) and TNF-α and down-regulation of IL-10 expression by infected macrophages. GM-CSF in combination with IFN-γ promoted IL-6 production and further reduction of IL-10 synthesis. Comparison of TNF-α vs. IL-10 and IL-6 vs. IL-10 ratios revealed the following hierarchy in regard to the pro-inflammatory potential of human macrophages infected with P. aeruginosa: untreated < treated with GM-CSF < treated with IFN-γ < treated with GM-CSF and IFN-γ

Increased CD8+ T cell responses to apoptotic T cell-associated antigens in multiple sclerosis.

BACKGROUND: Here, we evaluated the hypothesis that CD8(+) T cell responses to caspase-cleaved antigens derived from effector T cells undergoing apoptosis, may contribute to multiple sclerosis (MS) immunopathology. METHODS: The percentage of autoreactive CD8(+) T effector cells specific for various apoptotic T cell-associated self-epitopes (apoptotic epitopes) were detected in the peripheral blood and cerebrospinal fluid (CSF) by both enzyme-linked immunospot and dextramers of class I molecules complexed with relevant apoptotic epitopes. Moreover, the capacity of dextramer(+) CD8(+) T cells to produce interferon (IFN)-γ and/or interleukin (IL)-17 in response to the relevant apoptotic epitopes was evaluated by the intracellular cytokine staining. Cross-presentation assay of apoptotic T cells by dendritic cells was also evaluated ex vivo. RESULTS: We found that polyfunctional (IFN-γ and/or IL-17 producing) autoreactive CD8(+) T cells specific for apoptotic epitopes were represented in MS patients with frequencies significantly higher than in healthy donors. These autoreactive CD8(+) T cells with a strong potential to produce IFN-γ or IL-17 in response to the relevant apoptotic epitopes were significantly accumulated in the CSF from the same patients. In addition, the frequencies of these autoreactive CD8(+) T cells correlated with the disease disability. Cross-presentation assay revealed that caspase-cleaved cellular proteins are required to activate apoptotic epitope-specific CD8(+) T cells ex vivo. CONCLUSION: Taken together, these data indicate that apoptotic epitope-specific CD8(+) T cells with strong inflammatory potential are recruited at the level of the inflammatory site, where they may be involved in MS immunopathology through the production of high levels of inflammatory cytokines

Comparative Study on the Therapeutic Potential of Neurally Differentiated Stem Cells in a Mouse Model of Multiple Sclerosis

Background: Transplantation of neural stem cells (NSCs) is a promising novel approach to the treatment of neuroinflammatory diseases such as multiple sclerosis (MS). NSCs can be derived from primary central nervous system (CNS) tissue or obtained by neural differentiation of embryonic stem (ES) cells, the latter having the advantage of readily providing an unlimited number of cells for therapeutic purposes. Using a mouse model of MS, we evaluated the therapeutic potential of NSCs derived from ES cells by two different neural differentiation protocols that utilized adherent culture conditions and compared their effect to primary NSCs derived from the subventricular zone (SVZ). Methodology/Principal Findings: The proliferation and secretion of pro-inflammatory cytokines by antigen-stimulated splenocytes was reduced in the presence of SVZ-NSCs, while ES cell-derived NSCs exerted differential immunosuppressive effects. Surprisingly, intravenously injected NSCs displayed no significant therapeutic impact on clinical and pathological disease outcomes in mice with experimental autoimmune encephalomyelitis (EAE) induced by recombinant myelin oligodendrocyte glycoprotein, independent of the cell source. Studies tracking the biodistribution of transplanted ES cellderived NSCs revealed that these cells were unable to traffic to the CNS or peripheral lymphoid tissues, consistent with the lack of cell surface homing molecules. Attenuation of peripheral immune responses could only be achieved through multiple high doses of NSCs administered intraperitoneally, which led to some neuroprotective effects within the CNS

GM-CSF drives dysregulated hematopoietic stem cell activity and pathogenic extramedullary myelopoiesis in experimental spondyloarthritis

Dysregulated hematopoiesis occurs in several chronic inflammatory diseases, but it remains unclear how hematopoietic stem cells (HSCs) in the bone marrow (BM) sense peripheral inflammation and contribute to tissue damage in arthritis. Here, we show the HSC gene expression program is biased toward myelopoiesis and differentiation skewed toward granulocyte-monocyte progenitors (GMP) during joint and intestinal inflammation in experimental spondyloarthritis (SpA). GM-CSF-receptor is increased on HSCs and multipotent progenitors, favoring a striking increase in myelopoiesis at the earliest hematopoietic stages. GMP accumulate in the BM in SpA and, unexpectedly, at extramedullary sites: in the inflamed joints and spleen. Furthermore, we show that GM-CSF promotes extramedullary myelopoiesis, tissue-toxic neutrophil accumulation in target organs, and GM-CSF prophylactic or therapeutic blockade substantially decreases SpA severity. Surprisingly, besides CD4+ T cells and innate lymphoid cells, mast cells are a source of GM-CSF in this model, and its pathogenic production is promoted by the alarmin IL-33

Increased Systemic Th17 Cytokines Are Associated with Diastolic Dysfunction in Children and Adolescents with Diabetic Ketoacidosis

Diastolic dysfunction suggestive of diabetic cardiomyopathy is established in children with T1DM, but its pathogenesis is not well understood. We studied the relationships of systemic inflammatory cytokines/chemokines and cardiac function in 17 children with T1DM during and after correction of diabetic ketoacidosis (DKA). Twenty seven of the 39 measured cytokines/chemokines were elevated at 6–12 hours into treatment of DKA compared to values after DKA resolution. Eight patients displayed at least one parameter of diastolic abnormality (DA) during acute DKA. Significant associations were present between nine of the cytokine/chemokine levels and the DA over time. Interestingly, four of these nine interactive cytokines (GM-CSF, G-CSF, IL-12p40, IL-17) are associated with a Th17 mediated cell response. Both the DA and CCL7 and IL-12p40, had independent associations with African American patients. Thus, we report occurrence of a systemic inflammatory response and the presence of cardiac diastolic dysfunction in a subset of young T1DM patients during acute DKA

Sex Bias in Pathogenesis of Autoimmune Neuroinflammation: Relevance for Dimethyl Fumarate Immunomodulatory/Anti-oxidant Action

In the present study, upon showing sexual dimorphism in dimethyl fumarate (DMF) efficacy to moderate the clinical severity of experimental autoimmune encephalomyelitis (EAE) in Dark Agouti rats, cellular and molecular substrate of this dimorphism was explored. In rats of both sexes, DMF administration from the day of immunization attenuated EAE severity, but this effect was more prominent in males leading to loss of the sexual dimorphism observed in vehicle-administered controls. Consistently, in male rats, DMF was more efficient in diminishing the number of CD4+ T lymphocytes infiltrating spinal cord (SC) and their reactivation, the number of IL-17+ T lymphocytes and particularly cellularity of their highly pathogenic IFN-gamma+GM-CSF+IL-17+ subset. This was linked with changes in SC CD11b+CD45+TCR alpha beta- microglia/proinflammatory monocyte progeny, substantiated in a more prominent increase in the frequency of anti-inflammatory phygocyting CD163+ cells and the cells expressing high surface levels of immunoregulatory CD83 molecule (associated with apoptotic cells phagocytosis and implicated in downregulation of CD4+ T lymphocyte reactivation) among CD11b+CD45+TCR alpha beta- cells in male rat SC. These changes were associated with greater increase in the nuclear factor (erythroid-derived 2)-like 2 expression in male rats administered with DMF. In accordance with the previous findings, DMF diminished reactive nitrogen and oxygen species generation and consistently, SC level of advanced oxidation protein products, to the greater extent in male rats. Overall, our study indicates sex-specificity in the sensitivity of DMF cellular and molecular targets and encourages sex-based clinical research to define significance of sex for action of therapeutic agents moderating autoimmune neuroinflammation-/oxidative stress-related nervous tissue damage

JunB is essential for IL-23-dependent pathogenicity of Th17 cells

CD4+ T-helper cells producing interleukin-17 (IL-17), known as T-helper 17 (TH17) cells, comprise heterogeneous subsets that exhibit distinct pathogenicity. Although pathogenic and non-pathogenic TH17 subsets share a common RORγt-dependent TH17 transcriptional programme, transcriptional regulatory mechanisms specific to each of these subsets are mostly unknown. Here we show that the AP-1 transcription factor JunB is critical for TH17 pathogenicity. JunB, which is induced by IL-6, is essential for expression of RORγt and IL-23 receptor by facilitating DNA binding of BATF at the Rorc locus in IL-23-dependent pathogenic TH17 cells, but not in TGF-β1-dependent non-pathogenic TH17 cells. Junb-deficient T cells fail to induce TH17-mediated autoimmune encephalomyelitis and colitis. However, JunB deficiency does not affect the abundance of gut-resident non-pathogenic TH17 cells. The selective requirement of JunB for IL-23-dependent TH17 pathogenicity suggests that the JunB-dependent pathway may be a therapeutic target for autoimmune diseases