Identification of new sensitive biomarkers for the in vivo response to interferon‐β treatment in multiple sclerosis using DNA‐array evaluation

Objective: Neutralizing antibodies (NAbs) occur in a proportion of multiple sclerosis (MS) patients treated with interferon (IFN)‐β. NAbs impair the effect of treatment. The biological effect of IFN‐β can be measured as the induction of the myxovirus resistance protein A (MxA) molecule. However, other markers could be more sensitive for evaluating the response to IFN‐β. We used DNA array analysis to identify genes that are strongly induced in blood cells by IFN‐β, and measured their expression in MS patients with different NAb levels. Methods: Gene expression was studied on DNA arrays in untreated patients, in NAb negative patients, and in MS patients with varying NAb levels 9–12 h and 36–48 h after IFN‐β administration. The expression of selected genes was measured by real‐time PCR. NAb levels were assessed by a cytopathic effect assay. Results: Several hundred genes were induced 9–12 h after an injection of IFN‐β. The molecules CXCL10, CCL2 and IFI27 were among the most strongly induced. Gene induction was generally much less pronounced after 36–48 h, but IFI27 remained strongly induced. The strong induction of these molecules and MxA was confirmed by real‐time PCR. Induction of MxA, CCL2, CXCL10 and IFI27 was reduced in patients with low NAb levels and lost in patients with intermediate/high NAb levels. Conclusion: We identify IFI27, CCL2 and CXCL10 as sensitive biomarkers for the response to IFN‐β. The expression of these markers adequately reflects bioactivity of IFN‐ß as documented by the decreased induction in low NAb‐positive patients and the lost induction in patients with moderate/high NAb levels.     

γδ T cells: The overlooked T‐cell subset in demyelinating disease

γδ T cells represent a small subpopulation of T cells expressing a restricted repertoire of T‐cell receptors and, unlike αβ T cells, function more as cells of the innate immune system. These cells are found in skin and mucosal sites as well as secondary lymphoid tissues and frequently act as first line of defense sentinels. γδ T cells have been implicated in the pathogenesis of demyelinating disease, although little was known regarding their trafficking and effector functions. In this Mini‐Review, we highlight recent studies demonstrating that γδ T cells migrate rapidly to the CNS during experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. γδ T‐cell trafficking to the CNS is independent of β₂‐integrins and occurs well before onset of clinical signs of disease, peaking early during the acute phase of disease. γδ T‐cell‐mediated production of inflammatory cytokines, including interferon‐γ and tumor necrosis factor‐α, appears critical for EAE development, suggesting that these cells may set the stage for activation of other subsets of infiltrating effector cells. These data suggest that γδ T cells or subsets of γδ T cells may represent a new therapeutic target in demeylinating disease. © 2009 Wiley‐Liss, Inc.     

Interferon γ- and interleukin-4-secreting cells in multiple sclerosis

Interleukin 4 (IL-4)- and interferon γ (IFNγ)-secreting peripheral blood cells were enumerated by immunospot assay in 13 multiple sclerosis (MS) patients during exacerbations, in 24 patients with progressive multiple sclerosis (CPMS), and in 20 controls. Cells that spontaneously secreted IFNγ were significantly higher in MS patients experiencing an attack (P<0.001) than in controls or in CPMS (P<0.04). IL-4-secreting cell numbers were elevated significantly and to a comparable extent in both MS groups compared to controls. Our finding of increased numbers of IFNγ-secreting cells is in keeping with prior work showing increased IFNγ levels in the circulation prior to and durinng MS attacks and increased release of IFNγ to the supernatant in bulk cultured blood cells from MS patients. What role an increase in IL-4-secreting cells might play in MS is unclear, but it could relate to immune system regulation. Following in vitro exposure to MBP, IFNγ-secreting cell number rose above levels observed in the absence of stimulation in controls and in both MS groups with the rise in acutely exacerbating MS patients being significantly greater than in controls. Our results provide further evidence for reactivity to MBP in MS, the significance of which in terms of pathogenesis remains clouded.     

Interferon β-lb reduces Interferon γ-induced antigen-presenting capacity of human glial and B cells

Interferon (IFN) β-1b has been shown to alter the course of multiple sclerosis and to inhibit MHC class II expression, but its effect on antigen presentation has not been examined in a functional assay (Neurology 43 (1993) 655–661). The effect of IFNβ-1b on alloantigen presentation by human antigen-presenting cells (APC) including human fetal astrocytes (HFA) and microglia was examined. The effect of IFNβ-1b on the ability of B cells to present tetanus toxoid (TT) to TT-specific T cell lines was also examined. APC were pre-treated with IFNγ (100 units/ml), IFNβ-1b (10–2000 units/ml), or a combination of IFNγ and IFNβ-1b for 3 days and washed thoroughly prior to culture with allogeneic peripheral blood lymphocytes (PBL) for a period of 6 days. Lymphocyte proliferation was then measured by tritiated thymidine uptake. Treatment of the APC with IFNβ-1b resulted in a reduction in the IFNγ-enhanced alloantigen-induced T cell responses. This reduction ranged between 50 and 70%, was associated with a 30–50% reduction in HLA class II (DR) and 35–40% reduction in ICAM-1 expression on the HFA used as APC. IFNβ-1b pretreatment of B cells reduced their constitutive and IFNγ enhanced capacity to present TT to TT-specific T cell lines by 50–80%. This was associated with a 30 ± 11% mean reduction in class II (DR) expression and approximately 50 ± 1% reduction in ICAM-1 expression in IFNβ-1b + IFNγ-treated B cells compared to IFNγ-treated B cells (mean of three experiments). The reduction in antigen presentation and class II expression was not due to cellular toxicity as cell viability and tritiated thymidine uptake by APC were not altered significantly with treatment. The results suggest that IFNβ-1b can downregulate antigen presentation which could be related to altered expression of cell surface molecules on the APC and possibly also an effect on antigen processing.     

Interferon γ, interleukin 4 and transforming growth factor β in experimental autoimmune encephalomyelitis in lewis rats: Dynamics of cellular mrna expression in the central nervous system and lymphoid cells

The potential role of certain important immunoregulatory and effector cytokines in autoimmune neuroinflammation have been studied. We have examined the expression of mRNA, with in situ hybridization, of interferon -γ (IFN-γ), interleukin 4 (IL-4) and transforming growth factor β (TGF-β) both in sections of spinal cords and the antigen-induced expression of these cytokines by lymphoid cells after stimulation with a dominant encephalitogenic peptide of MBP (MBP 63–88) during the course of actively induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats. In spinal cords, the target organ in EAE, cells expressing mRNA for IFN-γ, first appeared at the onset of clinical signs, i.e., day 10 postimmunization (p.i.), peaked at the height of disease (day 13 p.i.), and then gradually decreased concomitant with recovery. Very few IL-4 mRNA-expressing cells appeared in the spinal cord with no clear relation to clinical signs or histopathology. In contrast, expression of mRNA for TGF-β did not increase until day 13 p.i., at height of the disease, shortly preceding recovery. These data are consistent with a disease upregulating role of IFN-γ, while TGF-β may act to limit central nervous system (CNS) inflammation. In lymphoid organs, primed MBP 63–88 reactive T cells showed an interesting time-dependent evolution of their cytokine production in vitro. Thus, early after immunization there was a conspicuous MBP 63–88-induced production of both IFN-γ and IL-4. Such cells may act in the initiation and promotion of the disease. Later, in the recovery phase, MBP 63–88 induced lymphoid cells to TGF-β production. Thus, an autoantigen-specific production of TGF-β occurred during EAE and hypothetically such a mechanism may serve to downregulate aggressive autoimmunity systemically. © 1995 Wiley-Liss, Inc.     

T cell receptor Vβ gene usage in the recognition of myelin basic protein by cerebrospinal fluid- and blood-derived T cells from patients with multiple sclerosis

Because of its proximity to the central nervous system, the cerebrospinal fluid (CSF) represents an important source of T cells that potentially could mediate putative autoimmune diseases such as multiple sclerosis (MS). To overcome the low CSF cellularity, we evaluated culture conditions that could expand CSF T cells, with a focus on the expression of T-cell receptor Vβ genes utilized by T cells specific for the potentially encephalitogenic autoantigen myelin basic protein (BP). Expansion of “activated” CSF cells with IL-2/IL-4 plus accessory cells optimally retained BP-responsive T cells that over-expressed Vβ1, Vβ2, Vβ;5, or Vβ;18, compared to expansion using supernatants from PHA-stimulated blood cells, or anti-CD3 antibody that led to different V gene bias and rare reactivity to BP. Sequential evaluation of paired CSF and blood samples from a relapsing remitting MS patient indicated that BP-reactive T cells were present in CSF during the period of clinical activity, and the pattern of BP recognition in CSF was partially reflected in blood, even after CSF reactivity had dissipated during remission. Over-expressed Vβ genes were not always constant, however, since in three sequential evaluations of a chronic progressive MS patient, Vβ genes over-expressed in the first BP-reactive CSF switched to a different Vβ gene bias that was present in the second and third CSF samples. Blood samples reflected each pattern of CSF Vβ gene bias, but retained the initial bias for at least 4 months after its disappearance from CSF. These data indicate that selective expansion of IL-2/Il-4-responsive CSF cells favors growth of the BP-reactive subpopulation, and, in a limited number of patients studied, reflected clinical disease activity. In comparison, blood T cells provided a partial but longer lasting reflection of the CSF BP reactivity and Vβ gene bias. © 1994 Wiley-Liss, Inc.     

α‐Synuclein activates innate immunity but suppresses interferon‐γ expression in murine astrocytes

Glial activation and neuroinflammation contribute to pathogenesis of neurodegenerative diseases, linked to neuron loss and dysfunction. α‐Synuclein (α‐syn), as a metabolite of neuron, can induce microglia activation to trigger innate immune response. However, whether α‐syn, as well as its mutants (A53T, A30P, and E46K), induces astrocyte activation and inflammatory response is not fully elucidated. In this study, we used A53T mutant and wild‐type α‐syns to stimulate primary astrocytes in dose‐ and time‐dependent manners (0.5, 2, 8, and 20 μg/ml for 24 hr or 3, 12, 24, and 48 hr at 2 μg/ml), and evaluated activation of several canonical inflammatory pathway components. The results showed that A53T mutant or wild‐type α‐syn significantly upregulated mRNA expression of toll‐like receptor (TLR)2, TLR3, nuclear factor‐κB and interleukin (IL)‐1β, displaying a pattern of positive dose–effect correlation or negative time–effect correlation. Such upregulation was confirmed at protein levels of TLR2 (at 20 μg/ml), TLR3 (at most doses), and IL‐1β (at 3 hr) by western blotting. Blockage of TLR2 other than TLR4 inhibited TLR3 and IL‐1β mRNA expressions. By contrast, interferon (IFN)‐γ was significantly downregulated at mRNA, protein, and protein release levels, especially at high concentrations of α‐syns or early time‐points. These findings indicate that α‐syn was a TLRs‐mediated immunogenic agent (A53T mutant stronger than wild‐type α‐syn). The stimulation patterns suggest that persistent release and accumulation of α‐syn is required for the maintenance of innate immunity activation, and IFN‐γ expression inhibition by α‐syn suggests a novel immune molecule interaction mechanism underlying pathogenesis of neurodegenerative diseases.     

Attenuation of β‐amyloid‐induced tauopathy via activation of CK2α/SIRT1: Targeting for cilostazol

β‐Amyloid (Aβ) deposits and hyperphosphorylated tau aggregates are the chief hallmarks in the Alzheimer's disease (AD) brains, but the strategies for controlling these pathological events remain elusive. We hypothesized that CK2‐coupled SIRT1 activation stimulated by cilostazol suppresses tau acetylation (Ac‐tau) and tau phosphorylation (P‐tau) by inhibiting activation of P300 and GSK3β. Aβ was endogenously overproduced in N2a cells expressing human APP Swedish mutation (N2aSwe) by exposure to medium containing 1% fetal bovine serum for 24 hr. Increased Aβ accumulation was accompanied by increased Ac‐tau and P‐tau levels. Concomitantly, these cells showed increased P300 and GSK3β P‐Tyr216 expression; their expressions were significantly reduced by treatment with cilostazol (3–30 μM) and resveratrol (20 μM). Moreover, decreased expression of SIRT1 and its activity by Aβ were significantly reversed by cilostazol as by resveratrol. In addition, cilostazol strongly stimulated CK2α phosphorylation and its activity, and then stimulated SIRT1 phosphorylation. These effects were confirmed by using the pharmacological inhibitors KT5720 (1 μM, PKA inhibitor), TBCA (20 μM, inhibitor of CK2), and sirtinol (20 μM, SIRT1 inhibitor) as well as by SIRT1 gene silencing and overexpression techniques. In conclusion, increased cAMP‐dependent protein kinase‐linked CK2/SIRT1 expression by cilostazol can be a therapeutic strategy to suppress the tau‐related neurodegeneration in the AD brain. © 2013 Wiley Periodicals, Inc.     

Glycogen synthase kinase‐3β phosphorylates synphilin‐1 in vitro

α‐Synuclein is known to be a major component of Lewy bodies and glial cytoplasmic inclusions in the brains of patients with α‐synucleinopathies. Synphilin‐1, an α‐synuclein‐associated protein, is also present in these inclusions. However, little is known about the post‐translational modifications of synphilin‐1. In the present study, it is reported that synphilin‐1 is phosphorylated by glycogen synthase kinase‐3βin vitro. It is well known that protein phosphorylation is involved in various physiological phenomena, including signal transduction and protein degradation. Therefore, phosphorylation of synphilin‐1 may play an important role in the function of this protein in the brain.     

Implication of Akt‐dependent Prp19α/14‐3‐3β/Cdc5L complex formation in neuronal differentiation

PRP19α and CDC5L are major components of the active spliceosome. However, their association process is still unknown. Here, we demonstrated that PRP19α/14‐3‐3β/CDC5L complex formation is regulated by Akt during nerve growth factor (NGF)‐induced neuronal differentiation of PC12 cells. Analysis of PRP19α mutants revealed that the phosphorylation of PRP19α at Thr 193 by Akt was critical for its binding with 14‐3‐3β to translocate into the nuclei and for PRP19α/14‐3‐3β/CDC5L complex formation in neuronal differentiation. Forced expression of either sense PRP19α or sense 14‐3‐3β RNAs promoted NGF‐induced neuronal differentiation, whereas down‐regulation of these mRNAs showed a suppressive effect. The nonphosphorylation mutant PRP19αT193A lost its binding ability with 14‐3‐3β and acted as a dominant‐negative mutant in neuronal differentiation. These results imply that Akt‐dependent phosphorylation of PRP19α at Thr193 triggers PRP19α/14‐3‐3β/CDC5L complex formation in the nuclei, likely to assemble the active spliceosome against neurogenic pre‐mRNAs. © 2010 Wiley‐Liss, Inc.     

δ‐Catenin/NPRAP: A new member of the glycogen synthase kinase‐3β signaling complex that promotes β‐catenin turnover in neurons

Through a multiprotein complex, glycogen synthase kinase‐3β (GSK‐3β) phosphorylates and destabilizes β‐catenin, an important signaling event for neuronal growth and proper synaptic function. δ‐Catenin, or NPRAP (CTNND2), is a neural enriched member of the β‐catenin superfamily and is also known to modulate neurite outgrowth and synaptic activity. In this study, we investigated the possibility that δ‐catenin expression is also affected by GSK‐3β signaling and participates in the molecular complex regulating β‐catenin turnover in neurons. Immunofluorescent light microscopy revealed colocalization of δ‐catenin with members of the molecular destruction complex: GSK‐3β, β‐catenin, and adenomatous polyposis coli proteins in rat primary neurons. GSK‐3β formed a complex with δ‐catenin, and its inhibition resulted in increased δ‐catenin and β‐catenin expression levels. LY294002 and amyloid peptide, known activators of GSK‐3β signaling, reduced δ‐catenin expression levels. Furthermore, δ‐catenin immunoreactivity increased and protein turnover decreased when neurons were treated with proteasome inhibitors, suggesting that the stability of δ‐catenin, like that of β‐catenin, is regulated by proteasome‐mediated degradation. Coimmunoprecipitation experiments showed that δ‐catenin overexpression promoted GSK‐3β and β‐catenin interactions. Primary cortical neurons and PC12 cells expressing δ‐catenin treated with proteasome inhibitors showed increased ubiquitinated β‐catenin forms. Consistent with the hypothesis that δ‐catenin promotes the interaction of the destruction complex molecules, cycloheximide treatment of cells overexpressing δ‐catenin showed enhanced β‐catenin turnover. These studies identify δ‐catenin as a new member of the GSK‐3β signaling pathway and further suggest that δ‐catenin is potentially involved in facilitating the interaction, ubiquitination, and subsequent turnover of β‐catenin in neuronal cells. © 2010 Wiley‐Liss, Inc.