Pro-inflammatory T helper 17 directly harms oligodendrocytes in neuroinflammation.

T helper (Th)17 cells are considered to contribute to inflammatory mechanisms in diseases such as multiple sclerosis (MS). However, the discussion persists regarding their true role in patients. Here, we visualized central nervous system (CNS) inflammatory processes in models of MS live in vivo and in MS brains and discovered that CNS-infiltrating Th17 cells form prolonged stable contact with oligodendrocytes. Strikingly, compared to Th2 cells, direct contact with Th17 worsened experimental demyelination, caused damage to human oligodendrocyte processes, and increased cell death. Importantly, we found that in comparison to Th2 cells, both human and murine Th17 cells express higher levels of the integrin CD29, which is linked to glutamate release pathways. Of note, contact of human Th17 cells with oligodendrocytes triggered release of glutamate, which induced cell stress and changes in biosynthesis of cholesterol and lipids, as revealed by single-cell RNA-sequencing analysis. Finally, exposure to glutamate decreased myelination, whereas blockade of CD29 preserved oligodendrocyte processes from Th17-mediated injury. Our data provide evidence for the direct and deleterious attack of Th17 cells on the myelin compartment and show the potential for therapeutic opportunities in MS.

CNS-localized myeloid cells capture living invading T cells during neuroinflammation.

To study the role of myeloid cells in the central nervous system (CNS) in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), we used intravital microscopy, assessing local cellular interactions in vivo in EAE animals and ex vivo in organotypic hippocampal slice cultures. We discovered that myeloid cells actively engulf invading living Th17 lymphocytes, a process mediated by expression of activation-dependent lectin and its T cell-binding partner, N-acetyl-D-glucosamine (GlcNAc). Stable engulfment resulted in the death of the engulfed cells, and, remarkably, enhancement of GlcNAc exposure on T cells in the CNS ameliorated clinical EAE symptoms. These findings demonstrate the ability of myeloid cells to directly react to pathogenic T cell infiltration by engulfing living T cells. Amelioration of EAE via GlcNAc treatment suggests a novel first-defense pathway of myeloid cells as an initial response to CNS invasion and demonstrates that T cell engulfment by myeloid cells can be therapeutically exploited in vivo.

Targeting CD52 does not affect murine neuron and microglia function.

The humanized anti-CD52 antibody alemtuzumab is successfully used in the treatment of multiple sclerosis (MS) and is thought to exert most of its therapeutic action by depletion and repopulation of mainly B and T lymphocytes. Although neuroprotective effects of alemtuzumab have been suggested, direct effects of anti-CD52 treatment on glial cells and neurons within the CNS itself have not been investigated so far. Here, we show CD52 expression in murine neurons, astrocytes and microglia, both in vitro and in vivo. As expected, anti CD52-treatment caused profound lymphopenia and improved disease symptoms in mice subjected to experimental autoimmune encephalomyelitis (EAE). CD52 blockade also had a significant effect on microglial morphology in organotypic hippocampal slice cultures but did not affect microglial functions. Furthermore, anti-CD52 neither changed baseline neuronal calcium, nor did it act neuroprotective in excitotoxicity models. Altogether, our findings argue against a functionally significant role of CD52 blockade on CNS neurons and microglia. The beneficial effects of alemtuzumab in MS may be exclusively mediated by peripheral immune mechanisms.

ß1-Integrin- and KV1.3 channel-dependent signaling stimulates glutamate release from Th17 cells.

Although the impact of Th17 cells on autoimmunity is undisputable, their pathogenic effector mechanism is still enigmatic. We discovered soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex proteins in Th17 cells that enable a vesicular glutamate release pathway that induces local intracytoplasmic calcium release and subsequent damage in neurons. This pathway is glutamine dependent and triggered by binding of ß1-integrin to vascular cell adhesion molecule 1 (VCAM-1) on neurons in the inflammatory context. Glutamate secretion could be blocked by inhibiting either glutaminase or KV1.3 channels, which are known to be linked to integrin expression and highly expressed on stimulated T cells. Although KV1.3 is not expressed in CNS tissue, intrathecal administration of a KV1.3 channel blocker or a glutaminase inhibitor ameliorated disability in experimental neuroinflammation. In humans, T cells from patients with multiple sclerosis secreted higher levels of glutamate, and cerebrospinal fluid glutamine levels were increased. Altogether, our findings demonstrate that ß1-integrin- and KV1.3 channel-dependent signaling stimulates glutamate release from Th17 cells upon direct cell-cell contact between Th17 cells and neurons.

Activation of microglia synergistically enhances neurodegeneration caused by MPP+ in human SH-SY5Y cells.

While MPP+ may not directly activate microglia, the initial neuronal damage inflicted by the toxin may trigger microglia, possibly leading to synergistic pro-apoptotic interaction between neuro-inflammation and toxin-induced neurotoxicity, which may further aggravate neurodegeneration. However, what molecular targets are synergistically up or downregulated during this interaction is not well understood. Here, we addressed this by co-culturing fully differentiated human SH-SY5Y cells treated with parkinsonian toxin 1-Methyl-4-phenylpyridinium (MPP+), with endotoxin-activated microglial cell line EOC 20 to determine how this interaction affects pro-apoptotic (p38, JNK, and bax:bcl2 ratios) and pro-survival (NF-κB, MEK1) signaling at both mRNA and protein levels. Concurrent MPP+ and endotoxin-treatment aggravated a decrease in SH-SY5Y cell viability and caused strong synergistic increases in the bax:bcl2 ratio, but also NF-κB and JNK signaling. These effects were attenuated by microglia inhibitor minocycline. Altogether, these data provide further molecular insights into the important role or even conditional requirement of microglia activation in the progressive neurodegenerative nature of PD.

Publisher Correction: Maladaptive cortical hyperactivity upon recovery from experimental autoimmune encephalomyelitis.

In the version of this article initially published, Inigo Ruiz de Azua's name was miscategorized. His given name is Inigo and his surname is Ruiz de Azua. This has been corrected in the HTML coding.

Maladaptive cortical hyperactivity upon recovery from experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) patients exhibit neuropsychological symptoms in early disease despite the immune attack occurring predominantly in white matter and spinal cord. It is unclear why neurodegeneration may start early in the disease and is prominent in later stages. We assessed cortical microcircuit activity by employing spiking-specific two-photon Ca2+ imaging in proteolipid protein-immunized relapsing-remitting SJL/J mice in vivo. We identified the emergence of hyperactive cortical neurons in remission only, independent of direct immune-mediated damage and paralleled by elevated anxiety. High levels of neuronal activity were accompanied by increased caspase-3 expression. Cortical TNFα expression was mainly increased by excitatory neurons in remission; blockade with intraventricular infliximab restored AMPA spontaneous excitatory postsynaptic current frequencies, completely recovered normal neuronal network activity patterns and alleviated elevated anxiety. This suggests a dysregulation of cortical networks attempting to achieve functional compensation by synaptic plasticity mechanisms, indicating a link between immune attack and early start of neurodegeneration.

In vivo and in vitro effects of multiple sclerosis immunomodulatory therapeutics on glutamatergic excitotoxicity.

In multiple sclerosis (MS), a candidate downstream mechanism for neuronal injury is glutamate (Glu)-induced excitotoxicity, leading to toxic increases in intraneuronal Ca(2+) . Here, we used in vivo two-photon imaging in the brain of TN-XXL transgenic Ca(2+) reporter mice to test whether promising oral MS therapeutics, namely fingolimod, dimethyl fumarate, and their respective metabolites fingolimod-phosphate and monomethyl fumarate, can protect neurons against acute glutamatergic excitotoxic damage. We also assessed whether these drugs can protect against excitotoxicity in vitro using primary cortical neurons, and whether they can directly inhibit Glu release from pathogenic T-helper 17 lymphocytes. In vivo, direct and acute (1 h) administration of 100 mM Glu to the brainstem resulted in a rapid and significant up-regulation in neuronal Ca(2+) signaling as well as morphological excitotoxic changes that were attenuated by the NMDA-receptor antagonist MK801. Direct CNS administration of MS drugs prior to Glu significantly delayed or reduced, but did not prevent the neuronal Ca(2+) increase or morphological changes. In vitro, prolonged (24 h) treatment of primary neurons with the fumarates significantly protected against neurotoxicity induced by Glu as well as NMDA, similar to MK801. Furthermore, monomethyl fumerate significantly reduced Glu release from pathogenic T-helper 17 lymphocytes. Overall, these data suggest that MS drugs may mediate neuroprotection via excitotoxicity modulating effects. Evidence suggests MS pathogenesis may involve neuronal excitotoxicity, induced by local release of glutamate. However, current MS drugs, including dimethyl fumerate (DMF) and fingolimod (FTY720) are largely anti-inflammatory and not yet fully tested for their neuroprotective potential. Here, we show that the drugs, in particular DMF metabolite monomethyl fumerate (MMF), protect neurons by excitotoxicity modulating effects. Th17, T-helper 17.

Defense of Elevated Body Weight Setpoint in Diet-Induced Obese Rats on Low Energy Diet Is Mediated by Loss of Melanocortin Sensitivity in the Paraventricular Hypothalamic Nucleus.

Some animals and humans fed a high-energy diet (HED) are diet-resistant (DR), remaining as lean as individuals who were naïve to HED. Other individuals become obese during HED exposure and subsequently defend the obese weight (Diet-Induced Obesity- Defenders, DIO-D) even when subsequently maintained on a low-energy diet. We hypothesized that the body weight setpoint of the DIO-D phenotype resides in the hypothalamic paraventricular nucleus (PVN), where anorexigenic melanocortins, including melanotan II (MTII), increase presynaptic GABA release, and the orexigenic neuropeptide Y (NPY) inhibits it. After prolonged return to low-energy diet, GABA inputs to PVN neurons from DIO-D rats exhibited highly attenuated responses to MTII compared with those from DR and HED-naïve rats. In DIO-D rats, melanocortin-4 receptor expression was significantly reduced in dorsomedial hypothalamus, a major source of GABA input to PVN. Unlike melanocortin responses, NPY actions in PVN of DIO-D rats were unchanged, but were reduced in neurons of the ventromedial hypothalamic nucleus; in PVN of DR rats, NPY responses were paradoxically increased. MTII-sensitivity was restored in DIO-D rats by several weeks' refeeding with HED. The loss of melanocortin sensitivity restricted to PVN of DIO-D animals, and its restoration upon prolonged refeeding with HED suggest that their melanocortin systems retain the ability to up- and downregulate around their elevated body weight setpoint in response to longer-term changes in dietary energy density. These properties are consistent with a mechanism of body weight setpoint.

Enhanced inflammatory and T-helper-1 type responses but suppressed lymphocyte proliferation in patients with seasonal affective disorder and treated by light therapy.

BACKGROUND: Animals show seasonal changes in the endocrine and immune system in response to winter stressors. Even though increased inflammation has been implicated in the pathophysiology of depression, whether immune disorder is a key mediator in seasonal affective depression (SAD) is unknown. Here, we hypothesized that short photoperiods in winter may induce inflammatory response, which contributes to SAD, and that light treatments should normalize immune function and improve depressive symptoms. METHODS: Twenty patients with a diagnosis of SAD, and a score on the HAM-29 of 20 or higher were recruited for this study. Twenty-one healthy subjects with no personal and family history of psychiatric disorder were matched to patients according to age and sex. Patients and controls were sampled during winter between November and January, inclusive. A subset of SAD patients (N=13) was re-sampled after 4 weeks of light therapy. Blood samples were assayed for macrophage activity, lymphocyte proliferation and cytokine release. RESULTS: SAD patients showed significantly higher macrophage activity and lower lymphocyte proliferation in winter compared to healthy subjects. The concentrations of macrophage-produced proinflammatory cytokines interleukin-1ß and tumour necrosis factor-α, and T-helper (Th)-1 produced cytokine, interferon-γ were all significantly increased. In contrast, no significant changes in Th2-produced cytokines were observed. Light therapy significantly improved depressive scores, which was associated with attenuation of decreased lymphocyte functions, increased macrophage activity and level of proinflammatory cytokines. CONCLUSION: SAD patients have increased macrophage and Th1 type responses in winter, and light therapy normalized immune functions and depressive symptoms. These results support an inflammatory hypothesis for SAD and an immunomodulatory role of light therapy.

FRET based ratiometric Ca(2+) imaging to investigate immune-mediated neuronal and axonal damage processes in experimental autoimmune encephalomyelitis.

BACKGROUND: Irreversible axonal and neuronal damage are the correlate of disability in patients suffering from multiple sclerosis (MS). A sustained increase of cytoplasmic free [Ca(2+)] is a common upstream event of many neuronal and axonal damage processes and could represent an early and potentially reversible step. NEW METHOD: We propose a method to specifically analyze the neurodegenerative aspects of experimental autoimmune encephalomyelitis by Förster Resonance Energy Transfer (FRET) imaging of neuronal and axonal Ca(2+) dynamics by two-photon laser scanning microscopy (TPLSM). RESULTS: Using the genetically encoded Ca(2+) sensor TN-XXL expressed in neurons and their corresponding axons, we confirm the increase of cytoplasmic free [Ca(2+)] in axons and neurons of autoimmune inflammatory lesions compared to those in non-inflamed brains. We show that these relative [Ca(2+)] increases were associated with immune-neuronal interactions. COMPARISON WITH EXISTING METHODS: In contrast to Ca(2+)-sensitive dyes the use of a genetically encoded Ca(2+) sensor allows reliable intraaxonal free [Ca(2+)] measurements in living anesthetized mice in health and disease. This method detects early axonal damage processes in contrast to e.g. cell/axon morphology analysis, that rather detects late signs of neurodegeneration. CONCLUSIONS: Thus, we describe a method to analyze and monitor early neuronal damage processes in the brain in vivo.

IL-17 and related cytokines involved in the pathology and immunotherapy of multiple sclerosis: Current and future developments.

Multiple sclerosis (MS), an autoimmune neurological disorder, is driven by self-reactive T helper (Th) cells. Research on the role of Th17 lymphocytes in MS pathogenesis has made significant progress in identifying various immunological as well as environmental factors that induce the differentiation and expansion of these cells, different subsets of Th17 cells with varying degrees of pathogenicity, and the role of the secreted effector cytokines. While approved therapies for MS offer significant benefit to patients, there remain unmet needs. Ongoing clinical trials aim to translate the advanced knowledge of Th17 cytokines to improved therapies. This review discusses the current status and future developments of research into the role of Th17 and related cytokines in MS pathogenesis and therapy.

ω-3 fatty acid eicosapentaenoic acid attenuates MPP+-induced neurodegeneration in fully differentiated human SH-SY5Y and primary mesencephalic cells.

Eicosapentaenoic acid (EPA), a neuroactive omega-3 fatty acid, has been demonstrated to exert neuroprotective effects in experimental models of Parkinson's disease (PD), but the cellular mechanisms of protection are unknown. Here, we studied the effects of EPA in fully differentiated human SH-SY5Y cells and primary mesencephalic neurons treated with MPP(+) . In both in-vitro models of PD, EPA attenuated an MPP(+) -induced reduction in cell viability. EPA also prevented the presence of electron-dense cytoplasmic inclusions in SH-SY5Y cells. Then, possible mechanisms of the neuroprotection were studied. In primary neurons, EPA attenuated an MPP(+) -induced increase in Tyrosine-related kinase B (TrkB) receptors. In SH-SY5Y cells, EPA down-regulated reactive oxygen species and nitric oxide. This antioxidant effect of EPA may have been mediated by its inhibition of neuronal NADPH oxidase and cyclo-oxygenase-2 (COX-2), as MPP(+) increased the expression of these enzymes. Furthermore, EPA prevented an increase in cytosolic phospholipase A2 (cPLA2), an enzyme linked with COX-2 in the potentially pro-inflammatory arachidonic acid cascade. Lastly, EPA attenuated an increase in the bax:bcl-2 ratio, and cytochrome c release. However, EPA did not prevent mitochondrial enlargement or a decrease in mitochondrial membrane potential. This study demonstrated cellular mechanisms by which EPA provided neuroprotective effects in experimental PD.

Cognitive enhancement by omega-3 fatty acids from child-hood to old age: findings from animal and clinical studies.

Omega-(n)-3 polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are major components of neuronal membranes and have a wide range of functions, from modulating synaptic plasticity and neurochemistry, to neuroimmune-modulation and neuroprotection. Thus, it is not surprising that n-3 PUFA are widely acknowledged to have cognitive-enhancing effects. Although clinical evidence is somewhat conflicting, probably in large part due to methodological issues, animal studies have consistently demonstrated that n-3 PUFA are indispensable for proper brain development, may enhance cognitive function in healthy, adult individuals and attenuate cognitive impairment in aging and age-related disorders, such as dementia. This review discusses and integrates up to date evidence from clinical and animal studies investigating the cognitive-enhancing effects of n-3 PUFA during development, child- and adult-hood, as well as old-age with associated neurodegenerative diseases, such as Alzheimer's disease. Furthermore, we cover the major underlying biochemical and neurophysiological mechanisms by which n-3 PUFA mediate these effects on cognition. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

Ethyl-eicosapentaenoate modulates changes in neurochemistry and brain lipids induced by parkinsonian neurotoxin 1-methyl-4-phenylpyridinium in mouse brain slices.

Evidence suggests a link between Parkinson's disease and the dietary intake of omega (n)-3 and n-6 polyunsaturated fatty acids (PUFAs). Presently, we investigated whether an acute dose of parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) affects brain n-3 and n-6 PUFA content and expression of fatty acid metabolic enzymes cytosolic phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX-2) in brain slices from C57Bl/6 mice. Furthermore, we investigated whether feeding a diet of n-3 PUFA ethyl-eicosapentaenoate (E-EPA) to these mice can attenuate the MPP(+) induced changes in brain PUFA content and expression of cPLA2 and COX-2, and attenuate MPP(+) induced changes in neurotransmitters and metabolites and apoptotic markers, bax, bcl-2 and caspase-3. MPP(+) increased brain content of n-6 PUFAs linoleic acid and arachidonic acid, and increased the mRNA expression of cPLA2. MPP(+) also depleted striatal dopamine levels and increased dopamine turnover, and depleted noradrenaline levels in the frontal cortex. The neurotoxin induced increases in bax, bcl-2 and caspase-3 mRNA expression that approached significance. E-EPA by itself increased brain n-3 content, including EPA and docosapentaenoic acid (C22:5, n-3), and increased cortical dopamine. More importantly, E-EPA attenuated the MPP(+) induced increase in n-6 fatty acids content, partially attenuated the striatal dopaminergic turnover, and prevented the increases of pro-apoptotic bax and caspase-3 mRNAs. In conclusion, increases in n-6 PUFAs in the acute stage of exposure to parkinsonian neurotoxins may promote pro-inflammatory conditions. EPA may provide modest beneficial effects in Parkinson's disease, but further investigation is warranted.

Behavior, neurotransmitters and inflammation in three regimens of the MPTP mouse model of Parkinson's disease.

Three common dosing regimens of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced model of Parkinson's disease (PD) were compared in C57BL/6 mice on behavior, striatal and extra-striatal neurotransmission, and brain cytokines, to clarify the differences between regimens on these variables. Acute regimen: Rotorod performance and open field grooming were decreased. Striatal dopamine (DA) was depleted, but DA turnover increased. Striatal noradrenalin (NA), frontal cortex serotonin (5-HT) and midbrain NA and DA were all depleted. Sub-acute regimen: Opposite to the acute regimen, rotorod and pole test performance, and open field grooming were all increased. Striatal DA was depleted, but DA turnover was increased more than in the acute regimen. Striatal 5-HT turnover and cortical NA were increased as well. Chronic regimen: Rotorod performance was impaired, but open field distance moved increased. Striatal DA was severely depleted and DA and 5-HT turnover strongly increased. Striatal 5-HT, frontal cortex NA and DA, and cortical DA were all depleted. Pro-inflammatory cytokines interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-10 were only increased in the chronic regimen, but these cytokines were found to be similarly related to striatal DA turnover in all regimens. The study demonstrated that the presence of behavioral differences between regimens may depend on the type of behavioral tests used and the extent to which dopaminergic, non-dopaminergic and extra-striatal neurotransmission are affected in the regimens. The study also provided additional evidence for the validity of the relatively new chronic MPTP/probenecid model. In all, the results suggested that dosing regimens should be carefully pre-considered.

Eicosapentaenoic acid (EPA) increases cell viability and expression of neurotrophin receptors in retinoic acid and brain-derived neurotrophic factor differentiated SH-SY5Y cells.

BACKGROUND: The n-3 polyunsaturated fatty acid, eicosapentaenoic acid (EPA) has been found to process neuroprotective effects. However, the exact cellular mechanisms are not well understood. Brain-derived neurotrophic factor (BDNF) is one of neurotrophins, which is involved in neuron differentiation, survival, and synaptogenesis. AIM OF THE STUDY: In this study, the potential neuroprotective effects of EPA, and its possible effects on BDNF and BDNF receptor expression were investigated in SH-SY5Y cells. METHODS: Both undifferentiated and retinoic acid (RA)-BDNF differentiated SH-SY5Y cells were treated with EPA and/or BDNF. The cell viability was determined by MTT assay. The expression of BDNF receptors, tyrosine kinase receptor B (TrkB) and p75(NTR) were tested by RT-PCR and Western blotting. RESULTS: In undifferentiated SH-SY5Y cells, either EPA or BDNF, or both did not affect the cell viability. In RA-BDNF differentiated SH-SY5Y cells, treatment with different doses of EPA (0.01, 0.1, 1.0, 10.0 microM) and BDNF (1 ng/ml) for 24 hours significantly increased the cell viability, while EPA or BDNF alone showed no effect. More importantly, RT-PCR and Western blotting results revealed that 24 hours treatment with EPA (0.01, 0.1, 1.0 microM) significantly increased the full-length TrkB (TrkB(TK+)), but not truncated TrkB (TrkB(TK-)) expression in these cells. An increase in p75(NTR) expression was also observed with 10.0 microM EPA treatment. Finally, co-incubation with either 100 nM staurosporine, a protein kinase inhibitor, or 500 nM K252a, a tyrosine kinase inhibitor completely abolished the EPA-induced increase in cell viability. CONCLUSIONS: Our results indicate that EPA exerts beneficial effects on cell survival through modulating neurotrophin receptor expression.

Effect of the COX-2 inhibitor celecoxib on behavioural and immune changes in an olfactory bulbectomised rat model of depression.

BACKGROUND: The olfactory bulbectomised (OBX) rat model is a chronic model of depression in which behavioural and neuroimmunoendocrine changes are reversed only after chronic antidepressant treatment. The cyclooxygenase 2 (COX-2) inhibitor celecoxib has been shown to improve the depressive symptoms in patients with major depression. METHODS: The association between blood and brain immunological and behavioural changes in chronic treatment with COX-2 inhibitor was explored in the OBX rats and their sham-operated controls. RESULTS: The OBX group showed significantly higher locomotor activity than the other groups in the first 5 min in the open field. In the home cage emergence test, the OBX group showed a significantly shorter latency period compared to the sham group (z = -3.192, p = 0.001) but there was no difference between the other three groups. In the hypothalamus, the OBX group had a significantly higher interleukin 1beta (IL-1beta) concentration than the OBX + celecoxib group (z = -1.89, p = 0.05) as well as a significantly higher IL-10 concentration (z = -1.995, p = 0.046). In the prefrontal cortex, the OBX group showed significantly higher concentrations of tumour necrosis factor alpha (z = -2.205, p = 0.028) and IL-1beta (z = -3.361, p = 0.001) than the OBX + celecoxib group, but a significantly lower concentration of IL-10 (p = -3.361, p = 0.001) than the OBX + celecoxib group. CONCLUSIONS: The results of this study supported the potential therapeutic role of the COX-2 inhibitor celecoxib. It is possible that the behavioural changes following the chronic administration of celecoxib to the OBX rats are associated with an attenuation of the increase in the pro-inflammatory cytokines in the brain.