Mood disturbances in newly diagnosed Parkinson's Disease patients reflect intrathecal inflammation.

In Parkinson's disease (PD), neuroinflammation may be involved in the pathogenesis of mood disorders, contributing to the clinical heterogeneity of the disease. The cerebrospinal fluid (CSF) levels of interleukin (IL)-1ß, IL-2, IL-6, IL-7, IL-8, IL-9, IL-12, IL-17, interferon (IFN)γ, macrophage inflammatory protein 1-alpha (MIP-1a), MIP-1b, granulocyte colony stimulating factor (GCSF), eotaxin, tumor necrosis factor (TNF), and monocyte chemoattractant protein 1 (MCP-1), were assessed in 45 newly diagnosed and untreated PD patients and in 44 control patients. Spearman's correlations were used to explore possible associations between CSF cytokines and clinical variables including mood. Benjamini-Hochberg (B-H) correction for multiple comparisons was applied. Linear regression was used to test significant associations correcting for other clinical variables. In PD patients, higher CSF concentrations of the inflammatory molecules IL-6, IL-9, IFNγ, and GCSF were found (all B-H corrected p < 0.02). Significant associations were found between BDI-II and the levels of IL-6 (Beta = 0.438; 95%CI 1.313-5.889; p = 0.003) and IL-8 (Beta = 0.471; 95%CI 0.185-0.743; p = 0.002). Positive associations were also observed between STAI-Y state and both IL-6 (Beta = 0.452; 95%CI 1.649-7.366; p = 0.003), and IL-12 (Beta = 0.417; 95%CI 2.238-13.379; p = 0.007), and between STAI-Y trait and IL-2 (Beta = 0.354; 95%CI 1.923-14.796; p = 0.012), IL-6 (Beta = 0.362; 95%CI 0.990-6.734; p = 0.01), IL-8 (Beta = 0.341; 95%CI 0.076-0.796; p = 0.019), IL-12 (Beta = 0.328; 95%CI 0.975-12.135; p = 0.023), and IL-17 (Beta = 0.334; 95CI 0.315-4.455; p = 0.025). An inflammatory CSF milieu may be associated with depression and anxiety in the early phases of PD, supporting a role of neuroinflammation in the pathogenesis of mood disturbances.

Extracellular vesicles from adipose mesenchymal stem cells target inflamed lymph nodes in experimental autoimmune encephalomyelitis.

BACKGROUND AIMS: Adipose mesenchymal stem cells (ASCs) represent a promising therapeutic approach in inflammatory neurological disorders, including multiple sclerosis (MS). Recent lines of evidence indicate that most biological activities of ASCs are mediated by the delivery of soluble factors enclosed in extracellular vesicles (EVs). Indeed, we have previously demonstrated that small EVs derived from ASCs (ASC-EVs) ameliorate experimental autoimmune encephalomyelitis (EAE), a murine model of MS. The precise mechanisms and molecular/cellular target of EVs during EAE are still unknown. METHODS: To investigate the homing of ASC-EVs, we intravenously injected small EVs loaded with ultra-small superparamagnetic iron oxide nanoparticles (USPIO) at disease onset in EAE-induced C57Bl/6J mice. Histochemical analysis and transmission electron microscopy were carried out 48 h after EV treatment. Moreover, to assess the cellular target of EVs, flow cytometry on cells extracted ex vivo from EAE mouse lymph nodes was performed. RESULTS: Histochemical and ultrastructural analysis showed the presence of labeled EVs in lymph nodes but not in lungs and spinal cord of EAE injected mice. Moreover, we identified the cellular target of EVs in EAE lymph nodes by flow cytometry: ASC-EVs were preferentially located in macrophages, with a consistent amount also noted in dendritic cells and CD4+ T lymphocytes. CONCLUSIONS: This represents the first direct evidence of the privileged localization of ASC-EVs in draining lymph nodes of EAE after systemic injection. These data provide prominent information on the distribution, uptake and retention of ASC-EVs, which may help in the development of EV-based therapy in MS.

Neural stem cell transplantation in patients with progressive multiple sclerosis: an open-label, phase 1 study.

Innovative pro-regenerative treatment strategies for progressive multiple sclerosis (PMS), combining neuroprotection and immunomodulation, represent an unmet need. Neural precursor cells (NPCs) transplanted in animal models of multiple sclerosis have shown preclinical efficacy by promoting neuroprotection and remyelination by releasing molecules sustaining trophic support and neural plasticity. Here we present the results of STEMS, a prospective, therapeutic exploratory, non-randomized, open-label, single-dose-finding phase 1 clinical trial ( NCT03269071 , EudraCT 2016-002020-86), performed at San Raffaele Hospital in Milan, Italy, evaluating the feasibility, safety and tolerability of intrathecally transplanted human fetal NPCs (hfNPCs) in 12 patients with PMS (with evidence of disease progression, Expanded Disability Status Scale ≥6.5, age 18-55 years, disease duration 2-20 years, without any alternative approved therapy). The safety primary outcome was reached, with no severe adverse reactions related to hfNPCs at 2-year follow-up, clearly demonstrating that hfNPC therapy in PMS is feasible, safe and tolerable. Exploratory secondary analyses showed a lower rate of brain atrophy in patients receiving the highest dosage of hfNPCs and increased cerebrospinal fluid levels of anti-inflammatory and neuroprotective molecules. Although preliminary, these results support the rationale and value of future clinical studies with the highest dose of hfNPCs in a larger cohort of patients.

Lower levels of glutathione in the anterior cingulate cortex associate with depressive symptoms and white matter hyperintensities in COVID-19 survivors.

SARS-CoV-2 is a novel coronavirus that mainly affects the respiratory system. However, clinical manifestations such as neurological symptoms, psychopathological outcomes and brain alterations suggest brain involvement during SARS-CoV-2 infection. Depressive symptoms and cerebral white matter hypodensities/hyperintensities (WMH) have been widely reported in COVID-19 survivors and have been shown to persist after recovery from infection. At the same time viral Infections, including COVID-19, have been shown to lead to oxidative stress. Glutathione (GSH) is the main antioxidant in the brain and reduced GSH levels have been implicated both in COVID-19 and depression. We therefore hypothesise that reduced GSH levels may be associated with depressive symptoms and WMH in COVID-19 survivors. Forty-nine participants (age 18-70) surviving COVID-19 underwent magnetic resonance imaging to measure WMH and brain GSH levels in the ACC, blood sampling to measure systemic inflammation and psychopathological assessment for depressive symptoms. ACC concentrations of GSH inversely associated with both depression scores and the number and volume of WMH. The volume of WMH also positively associated with depressive symptomatology. Finally, systemic inflammation negatively predicted GSH concentration in ACC. In conclusion, we observed overlapping associations of GSH levels in ACC, WMH and severity of depression in COVID-19 survivors, and confirmed the central role of systemic inflammation, thus warranting interest for further study of oxidative stress and antioxidants in the post-acute COVID-19 syndrome.

PD-1/PD-L Axis in Neuroinflammation: New Insights.

The approval of immune checkpoint inhibitors (ICIs) by the Food and Drug Administration (FDA) led to an improvement in the treatment of several types of cancer. The main targets of these drugs are cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein-1/programmed death-ligand 1 pathway (PD-1/PD-L1), which are important inhibitory molecules for the immune system. Besides being generally safer than common chemotherapy, the use of ICIs has been associated with several immune-related adverse effects (irAEs). Although rare, neurological adverse effects are reported within the irAEs in clinical trials, particularly in patients treated with anti-PD-1 antibodies or a combination of both anti-CTLA-4 and PD-1 drugs. The observations obtained from clinical trials suggest that the PD-1 axis may play a remarkable role in the regulation of neuroinflammation. Moreover, numerous studies in preclinical models have demonstrated the involvement of PD-1 in several neurological disorders. However, a comprehensive understanding of these cellular mechanisms remains elusive. Our review aims to summarize the most recent evidence concerning the regulation of neuroinflammation through PD-1/PD-L signaling, focusing on cell populations that are involved in this pathway.

Anti-SARS-CoV-2 T-stem cell memory persists in ocrelizumab-treated MS patients.

BACKGROUND: Development of long-lasting anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) T-cell responses in persons with multiple sclerosis (pwMS) treated with ocrelizumab is questioned. OBJECTIVE: Investigate antiviral T-cell responses after infection with SARS-CoV-2 in ocrelizumab-treated pwMS. Control groups included ocrelizumab-treated pwMS without SARS-CoV-2 infection, and non-MS individuals with and without SARS-CoV-2 infection. METHODS: Peripheral blood mononuclear cells were stimulated with SARS-CoV-2 peptide pools and T-cell reactivity was assessed by ELISPOT for interferon (IFN)-γ detection, and by multiparametric fluorescence-activated cell sorting (FACS) analyses for assessment and characterization of T-cell activation. RESULTS: ELISPOT assay against the spike and the N protein of SARS-CoV-2 displayed specific T-cell reactivity in 28/29 (96%) pwMS treated with ocrelizumab and infected by SARS-CoV-2, similar to infected persons without MS. This reactivity was present 1 year after infection and independent from the time of ocrelizumab infusion. FACS analysis following stimulation with SARS-CoV-2 peptide pools showed the presence of activation-induced markers (AIMs) in both CD4+ and CD8+ T-cell subsets in 96% and 92% of these individuals, respectively. Within naïve AIM+ CD4+ and CD8+ T-cells, we detected T memory stem cells, suggesting the acquisition of long-term memory. CONCLUSIONS: B-cell depletion using ocrelizumab does not impair the development of long-lasting anti-SARS-CoV-2 T-cell responses.

Interleukin 6 SNP rs1818879 Regulates Radiological and Inflammatory Activity in Multiple Sclerosis.

(1) Background: The clinical course of multiple sclerosis (MS) is critically influenced by the expression of different pro-inflammatory and anti-inflammatory cytokines. Interleukin 6 (IL-6) represents a major inflammatory molecule previously associated with exacerbated disease activity in relapsing remitting MS (RR-MS); however, the role of single-nucleotide polymorphisms (SNPs) in the IL-6 gene has not been fully elucidated in MS. (2) Methods: We explored in a cohort of 171 RR-MS patients, at the time of diagnosis, the associations between four IL-6 SNPs (rs1818879, rs1554606, rs1800797, and rs1474347), CSF inflammation, and clinical presentation. (3) Results: Using principal component analysis and logistic regression analysis we identified an association between rs1818879, radiological activity, and a set of cytokines, including the IL-1ß, IL-9, IL-10, and IL-13. No significant associations were found between other SNPs and clinical or inflammatory parameters. (4) Conclusions: The association between the rs1818879 polymorphism and subclinical neuroinflammatory activity suggests that interindividual differences in the IL-6 gene might influence the immune activation profile in MS.

Monocyte mitochondrial dysfunction, inflammaging, and inflammatory pyroptosis in major depression.

BACKGROUND: The macrophage theory of depression states that macrophages play an important role in Major Depressive Disorder (MDD). METHODS: MDD patients (N = 140) and healthy controls (N = 120) participated in a cross-sectional study investigating the expression of apoptosis/growth and lipid/cholesterol pathway genes (BAX, BCL10, EGR1, EGR2, HB-EGF, NR1H3, ABCA1, ABCG1, MVK, CD163, HMOX1) in monocytes (macrophage/microglia precursors). Gene expressions were correlated to a set of previously determined and reported inflammation-regulating genes and analyzed with respect to various clinical parameters. RESULTS: MDD monocytes showed an overexpression of the apoptosis/growth/cholesterol and the TNF genes forming an inter-correlating gene cluster (cluster 3) separate from the previously described inflammation-related gene clusters (containing IL1 and IL6). While upregulation of monocyte gene cluster 3 was a hallmark of monocytes of all MDD patients, upregulation of the inflammation-related clusters was confirmed to be found only in the monocytes of patients with childhood adversity. The latter group also showed a downregulation of the cholesterol metabolism gene MVK, which is known to play an important role in trained immunity and proneness to inflammation. CONCLUSIONS: The upregulation of cluster 3 genes in monocytes of all MDD patients suggests a premature aging of the cells, i.e. mitochondrial apoptotic dysfunction and TNF "inflammaging", as a general feature of MDD. The overexpression of the IL-1/IL-6 containing inflammation clusters and the downregulation of MVK in monocytes of patients with childhood adversity indicates a shift in this condition to a more severe inflammation form (pyroptosis) of the cells, additional to the signs of premature aging and inflammaging.

MYD88 L265P mutation and interleukin-10 detection in cerebrospinal fluid are highly specific discriminating markers in patients with primary central nervous system lymphoma: results from a prospective study.

Reliable biomarkers are needed to avoid diagnostic delay and its devastating effects in patients with primary central nervous system (CNS) lymphoma (PCNSL). We analysed the discriminating sensitivity and specificity of myeloid differentiation primary response (88) (MYD88) L265P mutation (mut-MYD88) and interleukin-10 (IL-10) in cerebrospinal fluid (CSF) of both patients with newly diagnosed (n = 36) and relapsed (n = 27) PCNSL and 162 controls (118 CNS disorders and 44 extra-CNS lymphomas). The concordance of MYD88 mutational status between tumour tissue and CSF sample and the source of ILs in PCNSL tissues were also investigated. Mut-MYD88 was assessed by TaqMan-based polymerase chain reaction. IL-6 and IL-10 messenger RNA (mRNA) was assessed on PCNSL biopsies using RNAscope technology. IL levels in CSF were assessed by enzyme-linked immunosorbent assay. Mut-MYD88 was detected in 15/17 (88%) PCNSL biopsies, with an 82% concordance in paired tissue-CSF samples. IL-10 mRNA was detected in lymphomatous B cells in most PCNSL; expression of IL-6 transcripts was negligible. In CSF samples, mut-MYD88 and high IL-10 levels were detected, respectively, in 72% and 88% of patients with newly diagnosed PCNSL and in 1% of controls; conversely, IL-6 showed a low discriminating sensitivity and specificity. Combined analysis of MYD88 and IL-10 exhibits a sensitivity and specificity to distinguish PCNSL of 94% and 98% respectively. Similar figures were recorded in patients with relapsed PCNSL. In conclusion, high detection rates of mut-MYD88 and IL-10 in CSF reflect, respectively, the MYD88 mutational status and synthesis of this IL in PCNSL tissue. These biomarkers exhibit a very high sensitivity and specificity in detecting PCNSL both at initial diagnosis and relapse. Implications of these findings in patients with lesions unsuitable for biopsy deserve to be investigated.

Higher baseline interleukin-1ß and TNF-α hamper antidepressant response in major depressive disorder.

Raised pro-inflammatory immune/inflammatory setpoints, leading to an increased production of peripheral cytokines, have been associated with Major Depressive Disorder (MDD) and with failure to respond to first-line antidepressant drugs. However, the usefulness of these biomarkers in clinical psychopharmacology has been questioned because single findings did not translate into the clinical practice, where patients are prescribed treatments upon clinical need. We studied a panel of 27 inflammatory biomarkers in a sample of 108 inpatients with MDD, treated with antidepressant monotherapy for 4 weeks upon clinical need in a specialized hospital setting, and assessed the predictive effect of baseline peripheral measures of inflammation on antidepressing efficacy (response rates and time-lagged pattern of decrease of depression severity) using a machine-learning approach with elastic net penalized regression, and multivariate analyses in the context of the general linear model. When considering both categorical and continuous measures of response, baseline levels of IL-1ß predicted non-response to antidepressants, with the predicted probability to respond being highly dispersed at low levels of IL-1ß, and stratifying toward non-response when IL-1ß is high. Significant negative effects were also detected for TNF-α, while IL-12 weakly predicted response. These findings support the usefulness of inflammatory biomarkers in the clinical psychopharmacology of depression, and add to ongoing research efforts aiming at defining reliable cutoff values to identify depressed patients in clinical settings with high inflammation, and low probability to respond.

A Single Nucleotide ADA Genetic Variant Is Associated to Central Inflammation and Clinical Presentation in MS: Implications for Cladribine Treatment.

In multiple sclerosis (MS), activated T and B lymphocytes and microglial cells release various proinflammatory cytokines, promoting neuroinflammation and negatively affecting the course of the disease. The immune response homeostasis is crucially regulated by the activity of the enzyme adenosine deaminase (ADA), as evidenced in patients with genetic ADA deficiency and in those treated with cladribine tablets. We investigated in a group of patients with MS the associations of a single nucleotide polymorphism (SNP) of ADA gene with disease characteristics and cerebrospinal fluid (CSF) inflammation. The SNP rs244072 of the ADA gene was determined in 561 patients with MS. Disease characteristics were assessed at the time of diagnosis; furthermore, in 258 patients, proinflammatory and anti-inflammatory molecules were measured in the CSF. We found a significant association between rs244072 and both clinical characteristics and central inflammation. In C-carriers, significantly enhanced disability and increased CSF levels of TNF, IL-5 and RANTES was observed. In addition, lower CSF levels of the anti-inflammatory cytokine IL-10 were found. Finally, the presence of the C allele was associated with a tendency of increased lymphocyte count. In MS patients, ADA SNP rs244072 is associated with CSF inflammation and disability. The selective targeting of the ADA pathway through cladribine tablet therapy could be effective in MS by acting on a pathogenically relevant biological mechanism.

Interleukin-6 Disrupts Synaptic Plasticity and Impairs Tissue Damage Compensation in Multiple Sclerosis.

Background: Synaptic plasticity helps in reducing the clinical expression of brain damage and represents a useful mechanism to compensate the negative impact of new brain lesions in multiple sclerosis (MS). Inflammation, altering synaptic plasticity, could negatively influence the disease course in relapsing-remitting MS (RR-MS). Objective: In the present study, we explored whether interleukin (IL)-6, a major proinflammatory cytokine involved in MS pathogenesis, alters synaptic plasticity and affects the ability to compensate for ongoing brain damage. Methods: The effect of IL-6 incubation on long-term potentiation (LTP) induction was explored in vitro, in mice hippocampal slices. We also explored the correlation between the cerebrospinal fluid (CSF) levels of this cytokine and the LTP-like effect induced by the paired associative stimulation (PAS) in a group of RR-MS patients. Finally, we examined the correlation between the CSF levels of IL-6 at the time of diagnosis and the prospective disease activity in a cohort of 150 RR-MS patients. Results:In vitro LTP induction was abolished by IL-6. Consistently, in patients with MS, a negative correlation emerged between IL-6 CSF concentrations and the effect of PAS. In MS patients, longer disease duration before diagnosis was associated with higher IL-6 CSF concentrations. In addition, elevated CSF levels of IL-6 were associated with greater clinical expression of new inflammatory brain lesions, unlike in patients with low or absent IL-6 concentrations, who had a better disease course. Conclusions: IL-6 interfering with synaptic plasticity mechanisms may impair the ability to compensate the clinical manifestation of new brain lesions in RR-MS patients.

Natural killer cells protect white matter integrity in bipolar disorder.

BACKGROUND: Bipolar Disorder (BD) associates with disrupted white matter (WM) microstructure and functional connectivity, and with a perturbation of the immune system. Higher cytokines, and reduced T cells, correlated with WM disruption and fMRI responses. A core component of the innate immune system, natural killer (NK) cells were detected in brain parenchyma, but never studied in BD. METHODS: We studied Diffusion Tensor Imaging (DTI) measures of water diffusion, fMRI corticolimbic functional response and connectivity, and multi-parameter cytofluorometry analysis of NK (CD56+) subpopulations, in 30 inpatients with active Bipolar Disorder type I. NK cells were also obtained in 36 healthy controls. RESULTS: Patients had significantly higher circulating counts of CD56+GMCSF+, CD56+INFγ+, and CD56+IL17+. NK cell levels positively associated to fractional anisotropy (FA) measures. CD56+TNFα+, CD56+INFγ+, and CD56+GMCSF+ directly correlated with FA, and inversely with radial (RD) and mean (MD) diffusivity. Duration of lithium treatment associated with higher CD56+TNFα+, CD56+IL2+, and CD56+IL4+, and positively associated with FA in tracts were NKs had significant effects. A mediation model suggested a partial mediation of CD56+TNFα+ cells, higher in patients on lithium, on the effects of lithium on FA. Frequencies of the same cytokine-producing NK cells also influenced fMRI cortico-limbic functional connectivity during processing of both, emotional and non-emotional stimuli. DISCUSSION: Higher circulating cytokine-producing NK cells associated with lithium treatment, and with DTI measures of WM integrity, partially mediating the effect of lithium on WM. The same cells associated with fMRI responses and connectivity, thus suggesting an effect on structural and functional connectomics in BD.

Transient Receptor Potential Vanilloid 1 Modulates Central Inflammation in Multiple Sclerosis.

Introduction: Disease course of multiple sclerosis (MS) is negatively influenced by proinflammatory molecules released by activated T and B lymphocytes and local immune cells. The endovanilloid system plays different physiological functions, and preclinical data suggest that transient receptor potential vanilloid type 1 (TRPV1) could modulate neuroinflammation in this disorder. Methods: The effect of TRPV1 activation on the release of two main proinflammatory cytokines, tumor necrosis factor (TNF) and interleukin (IL)-6, was explored in activated microglial cells. Furthermore, in a group of 132 MS patients, the association between the cerebrospinal fluid (CSF) levels of TNF and IL-6 and a single nucleotide polymorphisms (SNP) influencing TRPV1 protein expression and function (rs222747) was assessed. Results: In in vitro experiments, TRPV1 stimulation by capsaicin significantly reduced TNF and IL-6 release by activated microglial cells. Moreover, the anti-inflammatory effect of TRPV1 activation was confirmed by another TRPV1 agonist, the resiniferatoxin (RTX), whose effects were significantly inhibited by the TRPV1 antagonist, 5-iodoresiniferatoxin (5-IRTX). Vice versa, BV2 pre-treatment with 5-IRTX increased the inflammatory response induced by LPS. Moreover, in MS patients, a significant association emerged between TRPV1 SNP rs222747 and CSF TNF levels. In particular, the presence of a G allele, known to result in increased TRPV1 protein expression and function, was associated to lower CSF levels of TNF. Conclusions: Our results indicate that TRPV1 influences central inflammation in MS by regulating cytokine release by activated microglial cells. The modulation of the endovanilloid system may represent a useful approach to contrast neuroinflammation in MS.

Serum neurofilaments increase at progressive multifocal leukoencephalopathy onset in natalizumab-treated multiple sclerosis patients.

This study analyzed serum neurofilament light chains (NfL) in 2 European cohorts of 312 multiple sclerosis (MS) patients to investigate whether NfL are biomarkers of progressive multifocal leukoencephalopathy (PML) during natalizumab treatment. The cohort comprised 25 PML, 136 natalizumab-treated, and 151 untreated MS patients. Patients subsequently developing PML had similar NfL to other natalizumab-treated MS patients. At PML onset, NfL were 10-fold higher than in the pre-PML condition and in natalizumab-treated or untreated MS patients, and NfL continued to increase until onset of immune reconstitution inflammatory syndrome. The results suggest that in natalizumab-treated patients, NfL may represent an early and accessible marker of PML. Ann Neurol 2019;85:606-610.

AMBRA1 Controls Regulatory T-Cell Differentiation and Homeostasis Upstream of the FOXO3-FOXP3 Axis.

Regulatory T cells (Treg) are necessary to maintain immunological tolerance and are key players in the control of autoimmune disease susceptibility. Expression of the transcription factor FOXP3 is essential for differentiation of Treg cells and indispensable for their suppressive function. However, there is still a lack of knowledge about the mechanisms underlying its regulation. Here, we demonstrate that pro-autophagy protein AMBRA1 is also a key modulator of T cells, regulating the complex network that leads to human Treg differentiation and maintenance. Indeed, through its ability to interact with the phosphatase PP2A, AMBRA1 promotes the stability of the transcriptional activator FOXO3, which, in turn, triggers FOXP3 transcription. Furthermore, we found that AMBRA1 plays a significant role in vivo by regulating Treg cell induction in mouse models of both tumor growth and multiple sclerosis, thus highlighting the role of AMBRA1 in the control of immune homeostasis.

Delayed treatment of MS is associated with high CSF levels of IL-6 and IL-8 and worse future disease course.

BACKGROUND: Clinical deterioration of relapsing-remitting MS (RR-MS) patients reflects not only the number and severity of overt inflammatory and demyelinating episodes, but also subtle central damage caused by persistent exposure to inflammatory molecules. OBJECTIVE: To explore the correlation between levels of CSF inflammatory molecules at the time of diagnosis and both demographic and clinical characteristics of a large sample of RR-MS patients, as well as the predictive value of cytokine levels on their prospective disease course. METHODS: In 205 patients diagnosed with RR-MS, we measured at the time of diagnosis the CSF levels of inflammatory molecules. Clinical and MRI evaluation was collected at the time of CSF withdrawal and during a median follow-up of 3 years. RESULTS: The time interval between the first anamnestic episode of focal neurological dysfunction and RR-MS diagnosis was the main factor associated with high CSF levels of IL-6 and IL-8. Furthermore, elevated CSF levels of these cytokines correlated with enhanced risk of clinical and radiological disease reactivation, switch to second-line treatments, and with disability progression in the follow-up. CONCLUSIONS: Delayed diagnosis and treatment initiation are associated with higher CSF levels of IL-6 and IL-8 in RR-MS, leading to worsening disease course and poor response to treatments.

Calcitonin gene-related peptide decreases IL-1beta, IL-6 as well as Ym1, Arg1, CD163 expression in a brain tissue context-dependent manner while ameliorating experimental autoimmune encephalomyelitis.

Activation states of immune cells (among them, the so-called pro- or anti-inflammatory states) influence the pathogenesis of multiple sclerosis (MS). The neuropeptide calcitonin gene-related peptide (CGRP) can exert a pro- or anti-inflammatory role in a context-dependent manner. In mice CGRP was found to attenuate the development of experimental autoimmune encephalomyelitis (EAE, a common MS animal model). We analyzed CGRP effects on the expression of cytokines and markers of activation states, as well as its intracellular cascade, following intrathecal administration during EAE immunization. Real Time quantitative-PCR (RT-PCR) showed that IL-1beta and IL-6 (associated to a pro-inflammatory state in EAE), but also Ym1 (also known as Chil3), Arg1 and CD163 (associated to an anti-inflammatory state in EAE) were decreased in the encephalon (devoid of cerebellum). In the cerebellum itself, IL-1beta and Ym1 were decreased. TNF-alpha (associated to a pro-inflammatory state in EAE), but also IL-10 (associated to another type of anti-inflammatory state) and BDNF were unchanged in these two regions. No changes were detected in the spinal cord. Additional tendencies toward a change (as revealed by RT-PCR) were again decreases: IL-10 in the encephalon and Arg1 in the spinal cord. CGRP decreased percentage of Ym1+/CD68+ immunoreactive cells and cell density of infiltrates in the cervical spinal cord pia mater. Instead, Ym1 in the underlying parenchyma and at thoracic and lumbar levels, as well as Arg1, were unchanged. In cultured microglia the neuropeptide decreased Ym1, but not Arg1, immunoreactivity. Inducible NOS (iNOS) was unchanged in spinal cord microglia and astrocytes. The neuropeptide increased the activation of ERK1/2 in the astrocytes of the spinal cord and in culture, but did not influence the activation of ERK1/2 or p38 in the spinal cord microglia. Finally, in areas adjacent to infiltration sites CGRP-treated microglia showed a larger ramification radius. In conclusion, CGRP-induced EAE amelioration was associated to a concomitant, context-dependent decrease in the expression of markers belonging to both pro- or anti-inflammatory activation states of immune cells. It can be hypothesized that CGRP-induced EAE attenuation is obtained through a novel mechanism that promotes down-regulation of immune cell activation that facilitates the establishment of a beneficial environment in EAE provided possibly also by other factors.

Extracellular Vesicles Containing IL-4 Modulate Neuroinflammation in a Mouse Model of Multiple Sclerosis.

Extracellular vesicles (EVs) play a major role in cell-to-cell communication in physiological and pathological conditions, and their manipulation may represent a promising therapeutic strategy. Microglia, the parenchymal mononuclear phagocytes of the brain, modulate neighboring cells also through the release of EVs. The production of custom EVs filled with desired molecules, possibly targeted to make their uptake cell specific, and their administration in biological fluids may represent a valid approach for drug delivery. We engineered a murine microglia cell line, BV-2, to release EVs overexpressing the endogenous "eat me" signal Lactadherin (Mfg-e8) on the surface to target phagocytes and containing the anti-inflammatory cytokine IL-4. A single injection of 107 IL-4+Mfg-e8+ EVs into the cisterna magna modulated established neuroinflammation and significantly reduced clinical signs in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Injected IL-4+Mfg-e8+ EVs target mainly phagocytes (i.e., macrophages and microglia) surrounding liquoral spaces, and their cargo promote the upregulation of anti-inflammatory markers chitinase 3-like 3 (ym1) and arginase-1 (arg1), significantly reducing tissue damage. Engineered EVs may represent a biological drug delivery tool able to deliver multiple functional molecules simultaneously to treat neuroinflammatory diseases.