HNF4α, SP1 and c-myc are master regulators of CNS autoimmunity.

Hepatocyte nuclear factor 4 α (HNF4α), a transcription factor (TF) essential for embryonic development, has been recently shown to regulate the expression of inflammatory genes. To characterize HNF4a function in immunity, we measured the effect of HNF4α antagonists on immune cell responses in vitro and in vivo. HNF4α blockade reduced immune activation in vitro and disease severity in the experimental model of multiple sclerosis (MS). Network biology studies of human immune transcriptomes unraveled HNF4α together with SP1 and c-myc as master TF regulating differential expression at all MS stages. TF expression was boosted by immune cell activation, regulated by environmental MS risk factors and higher in MS immune cells compared to controls. Administration of compounds targeting TF expression or function demonstrated non-synergic, interdependent transcriptional control of CNS autoimmunity in vitro and in vivo. Collectively, we identified a coregulatory transcriptional network sustaining neuroinflammation and representing an attractive therapeutic target for MS and other inflammatory disorders.

Convergence between Microglia and Peripheral Macrophages Phenotype during Development and Neuroinflammation.

Differently from other myeloid cells, microglia derive exclusively from precursors originating within the yolk sac and migrate to the CNS under development, without any contribution from fetal liver or postnatal hematopoiesis. Consistent with their unique ontology, microglia may express specific physiological markers, which have been partly described in recent years. Here we wondered whether profiles distinguishing microglia from peripheral macrophages vary with age and under pathology. To this goal, we profiled transcriptomes of microglia throughout the lifespan and included a parallel comparison with peripheral macrophages under physiological and neuroinflammatory settings using age- and sex-matched wild-type and bone marrow chimera mouse models. This comprehensive approach demonstrated that the phenotypic differentiation between microglia and peripheral macrophages is age-dependent and that peripheral macrophages do express some of the most commonly described microglia-specific markers early during development, such as Fcrls, P2ry12, Tmem119, and Trem2. Further, during chronic neuroinflammation CNS-infiltrating macrophages and not peripheral myeloid cells acquire microglial markers, indicating that the CNS niche may instruct peripheral myeloid cells to gain the phenotype and, presumably, the function of the microglia cell. In conclusion, our data provide further evidence about the plasticity of the myeloid cell and suggest caution in the strict definition and application of microglia-specific markers.SIGNIFICANCE STATEMENT Understanding the respective role of microglia and infiltrating monocytes in neuroinflammatory conditions has recently seemed possible by the identification of a specific microglia signature. Here instead we provide evidence that peripheral macrophages may express some of the most commonly described microglia markers at some developmental stages or pathological conditions, in particular during chronic neuroinflammation. Further, our data support the hypothesis about phenotypic plasticity and convergence among distinct myeloid cells so that they may act as a functional unit rather than as different entities, boosting their mutual functions in different phases of disease. This holds relevant implications in the view of the growing use of myeloid cell therapies to treat brain disease in humans.

Cytokines Stimulate the Release of Microvesicles from Myeloid Cells Independently from the P2X7 Receptor/Acid Sphingomyelinase Pathway.

Microvesicles (MVs) are membrane particles of 200-500 nm released by all cell types constitutively. MVs of myeloid origin are found increased in the cerebrospinal fluid (CSF) of patients suffering from neuroinflammatory disorders, although the factors triggering their production have never been defined. Here, we report that both pro- and anti-inflammatory cytokines, specifically interferon-γ and interleukin-4, are equally able to stimulate the production of MVs from microglia cells and monocytes. Additionally, we found this process to be independent from the best characterized molecular pathway so far described for membrane shedding, which is centered on the purinergic receptor P2X7, whose activation by high concentrations of extracellular ATP (exATP) results in membrane blebbing operated by the secreted enzyme acid sphingomyelinase (ASMase). Moreover, a potent inhibitor of ASMase, injected in a mouse model of multiple sclerosis, failed to reduce the number of MVs in their CSF. This suggests that cytokines, rather than exATP, may exert a long-term control of MV production in the context of chronic inflammation, where both pro- and anti-inflammatory factors play coordinated roles.

Neural precursor cell-secreted TGF-ß2 redirects inflammatory monocyte-derived cells in CNS autoimmunity.

In multiple sclerosis, the pathological interaction between autoreactive Th cells and mononuclear phagocytes in the CNS drives initiation and maintenance of chronic neuroinflammation. Here, we found that intrathecal transplantation of neural stem/precursor cells (NPCs) in mice with experimental autoimmune encephalomyelitis (EAE) impairs the accumulation of inflammatory monocyte-derived cells (MCs) in the CNS, leading to improved clinical outcome. Secretion of IL-23, IL-1, and TNF-α, the cytokines required for terminal differentiation of Th cells, decreased in the CNS of NPC-treated mice, consequently inhibiting the induction of GM-CSF-producing pathogenic Th cells. In vivo and in vitro transcriptome analyses showed that NPC-secreted factors inhibit MC differentiation and activation, favoring the switch toward an antiinflammatory phenotype. Tgfb2-/- NPCs transplanted into EAE mice were ineffective in impairing MC accumulation within the CNS and failed to drive clinical improvement. Moreover, intrathecal delivery of TGF-ß2 during the effector phase of EAE ameliorated disease severity. Taken together, these observations identify TGF-ß2 as the crucial mediator of NPC immunomodulation. This study provides evidence that intrathecally transplanted NPCs interfere with the CNS-restricted inflammation of EAE by reprogramming infiltrating MCs into antiinflammatory myeloid cells via secretion of TGF-ß2.

Neural Stem Cell Transplantation Induces Stroke Recovery by Upregulating Glutamate Transporter GLT-1 in Astrocytes.

Ischemic stroke is the leading cause of disability, but effective therapies are currently widely lacking. Recovery from stroke is very much dependent on the possibility to develop treatments able to both halt the neurodegenerative process as well as to foster adaptive tissue plasticity. Here we show that ischemic mice treated with neural precursor cell (NPC) transplantation had on neurophysiological analysis, early after treatment, reduced presynaptic release of glutamate within the ipsilesional corticospinal tract (CST), and an enhanced NMDA-mediated excitatory transmission in the contralesional CST. Concurrently, NPC-treated mice displayed a reduced CST degeneration, increased axonal rewiring, and augmented dendritic arborization, resulting in long-term functional amelioration persisting up to 60 d after ischemia. The enhanced functional and structural plasticity relied on the capacity of transplanted NPCs to localize in the peri-ischemic and ischemic area, to promote the upregulation of the glial glutamate transporter 1 (GLT-1) on astrocytes and to reduce peri-ischemic extracellular glutamate. The upregulation of GLT-1 induced by transplanted NPCs was found to rely on the secretion of VEGF by NPCs. Blocking VEGF during the first week after stroke reduced GLT-1 upregulation as well as long-term behavioral recovery in NPC-treated mice. Our results show that NPC transplantation, by modulating the excitatory-inhibitory balance and stroke microenvironment, is a promising therapy to ameliorate disability, to promote tissue recovery and plasticity processes after stroke. SIGNIFICANCE STATEMENT: Tissue damage and loss of function occurring after stroke can be constrained by fostering plasticity processes of the brain. Over the past years, stem cell transplantation for repair of the CNS has received increasing interest, although underlying mechanism remain elusive. We here show that neural stem/precursor cell transplantation after ischemic stroke is able to foster axonal rewiring and dendritic plasticity and to induce long-term functional recovery. The observed therapeutic effect of neural precursor cells seems to underlie their capacity to upregulate the glial glutamate transporter on astrocytes through the vascular endothelial growth factor inducing favorable changes in the electrical and molecular stroke microenvironment. Cell-based approaches able to influence plasticity seem particularly suited to favor poststroke recovery.

Myeloid cells as target of fingolimod action in multiple sclerosis.

OBJECTIVE: To track the effects of fingolimod, an approved drug for multiple sclerosis (MS), on the activation of myeloid cells from the periphery to the CNS. METHODS: In vitro and ex vivo immunologic studies coupled with flow cytometry were performed to evaluate the action of fingolimod on lipopolysaccharide (LPS)-induced expression of activation markers in human monocytes from healthy participants, participants with untreated MS, and participants with fingolimod-treated MS. In vivo administration of fingolimod during experimental autoimmune encephalomyelitis (EAE) was established to verify the activation state of splenic, CNS infiltrating, and CNS resident myeloid cells ex vivo at flow cytometer. RESULTS: We found that in vitro exposure of human monocytes to fingolimod inhibited LPS-induced CD25 and CD150 expression and tumor necrosis factor-α (TNF-α) secretion without altering immune cell survival. Further, EAE treatment with fingolimod led to reduced amounts of TNF-α produced by myeloid cells in vivo in the spleen and CNS. Finally, while displaying normal induction of CD25 and CD150 levels at high LPS concentration, monocytes from patients with fingolimod-treated MS showed significantly higher activation threshold at suboptimal LPS stimulation than controls. CONCLUSIONS: The inhibition of myeloid cell activation may be part of the immunosuppressive action of fingolimod and take place in the periphery and in the CNS.

Transcript profiling of different types of multiple sclerosis lesions yields FGF1 as a promoter of remyelination.

Chronic demyelination is a pathological hallmark of multiple sclerosis (MS). Only a minority of MS lesions remyelinates completely. Enhancing remyelination is, therefore, a major aim of future MS therapies. Here we took a novel approach to identify factors that may inhibit or support endogenous remyelination in MS. We dissected remyelinated, demyelinated active, and demyelinated inactive white matter MS lesions, and compared transcript levels of myelination and inflammation-related genes using quantitative PCR on customized TaqMan Low Density Arrays. In remyelinated lesions, fibroblast growth factor (FGF) 1 was the most abundant of all analyzed myelination-regulating factors, showed a trend towards higher expression as compared to demyelinated lesions and was significantly higher than in control white matter. Two MS tissue blocks comprised lesions with adjacent de- and remyelinated areas and FGF1 expression was higher in the remyelinated rim compared to the demyelinated lesion core. In functional experiments, FGF1 accelerated developmental myelination in dissociated mixed cultures and promoted remyelination in slice cultures, whereas it decelerated differentiation of purified primary oligodendrocytes, suggesting that promotion of remyelination by FGF1 is based on an indirect mechanism. The analysis of human astrocyte responses to FGF1 by genome wide expression profiling showed that FGF1 induced the expression of the chemokine CXCL8 and leukemia inhibitory factor, two factors implicated in recruitment of oligodendrocytes and promotion of remyelination. Together, this study presents a transcript profiling of remyelinated MS lesions and identified FGF1 as a promoter of remyelination. Modulation of FGF family members might improve myelin repair in MS.

iPSC-derived neural precursors exert a neuroprotective role in immune-mediated demyelination via the secretion of LIF.

The possibility of generating neural stem/precursor cells (NPCs) from induced pluripotent stem cells (iPSCs) has opened a new avenue of research that might nurture bench-to-bedside translation of cell transplantation protocols in central nervous system myelin disorders. Here we show that mouse iPSC-derived NPCs (miPSC-NPCs)-when intrathecally transplanted after disease onset-ameliorate clinical and pathological features of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Transplanted miPSC-NPCs exert the neuroprotective effect not through cell replacement, but through the secretion of leukaemia inhibitory factor that promotes survival, differentiation and the remyelination capacity of both endogenous oligodendrocyte precursors and mature oligodendrocytes. The early preservation of tissue integrity limits blood-brain barrier damage and central nervous system infiltration of blood-borne encephalitogenic leukocytes, ultimately responsible for demyelination and axonal damage. While proposing a novel mechanism of action, our results further expand the therapeutic potential of NPCs derived from iPSCs in myelin disorders.

Autocrine and immune cell-derived BDNF in human skeletal muscle: implications for myogenesis and tissue regeneration.

The neurotrophin system has a role in skeletal muscle biology. Conditional depletion of BDNF in mouse muscle precursor cells alters myogenesis and regeneration in vivo. However, the expression, localization and function of BDNF in human skeletal muscle tissue is not known, so the relevance of the rodent findings for human muscle are unknown. Here we address this by combining ex vivo histological investigations on human biopsies with in vitro analyses of human primary myocytes. We found that BDNF was expressed by precursor and differentiated cells both in vitro and in vivo. Differential analysis of BDNF receptors showed expression of p75NTR and not of TrkB in myocytes, suggesting that the BDNF-p75NTR axis is predominant in human skeletal muscle cells. Several in vitro functional experiments demonstrated that BDNF gene silencing or protein blockade in myoblast cultures hampered myogenesis. Finally, histological investigations of inflammatory myopathy biopsies revealed that infiltrating immune cells localized preferentially near p75NTR-positive regenerating fibres and that they produced BDNF. In conclusion, BDNF is an autocrine factor for skeletal muscle cells and may regulate human myogenesis. Furthermore, the preferential localization of BDNF-producing immune cells near p75NTR-positive regenerating myofibres suggests that immune cell-derived BDNF may sustain tissue repair in inflamed muscle.

Human neurotrophin receptor p75NTR defines differentiation-oriented skeletal muscle precursor cells: implications for muscle regeneration.

Satellite cells are resident stem cells of adult skeletal muscle that have roles in tissue repair. Although several efforts have led to the functional characterization of distinct myogenic populations in animal models, the translation of these findings to humans has been limited. Here, we analyzed the expression and function of the neurotrophin receptor p75NTR in human skeletal muscle precursor cells. We combined histological investigations of muscle biopsies with molecular and cellular analyses of primary muscle precursor cells. p75NTR is expressed by most satellite cells in vivo and is a marker for regenerating fibers in inflamed and dystrophic muscle. p75NTR mRNA and protein are also detectable in primary myoblasts, and these levels increase transiently when cell differentiation is triggered. Transcriptome analyses of p75NTR high versus p75NTR low muscle cells showed that p75NTR is the prototype marker for a precursor cell population that has a broad transcriptional repertoire associated with muscle development and maturation. Several in vitro experiments, including receptor blockade and gene silencing in myoblasts, proved that p75NTR specifically regulates myogenesis and dystrophin expression. Taken together, the results indicate that p75NTR is a novel marker of human differentiation-prone muscle precursor cells that is involved in myogenesis in vivo and in vitro.

BDNF and its receptors in human myasthenic thymus: implications for cell fate in thymic pathology.

Here we show that in myasthenic thymus several cell types, including thymic epithelial cells (TEC) and immune cells, were the source and the target of the neurotrophic factor brain-derived growth factor (BDNF). Interestingly, many actively proliferating medullary thymocytes expressed the receptor TrkB in vivo in involuted thymus, while this population was lost in hyperplastic or neoplastic thymuses. Furthermore, in hyperplastic thymuses the robust coordinated expression of BDNF in the germinal centers together with the receptor p75NTR on all proliferating B cells strongly suggests that this factor regulates germinal center reaction. Finally, all TEC dying of apoptosis expressed BDNF receptors, indicating that this neurotrophin is involved in TEC turnover. In thymomas both BDNF production and receptor expression in TEC were strongly hindered. This may represent an attempt of tumour escape from cell death.

Chemokines in multiple sclerosis: CXCL12 and CXCL13 up-regulation is differentially linked to CNS immune cell recruitment.

Understanding the mechanisms of immune cell migration to multiple sclerosis lesions offers significant therapeutic potential. This study focused on the chemokines CXCL12 (SDF-1) and CXCL13 (BCA-1), both of which regulate B cell migration in lymphoid tissues. We report that immunohistologically CXCL12 was constitutively expressed in CNS parenchyma on blood vessel walls. In both active and chronic inactive multiple sclerosis lesions CXCL12 protein was elevated and detected on astrocytes and blood vessels. Quantitative PCR demonstrated that CXCL13 was produced in actively demyelinating multiple sclerosis lesions, but not in chronic inactive lesions or in the CNS of subjects who had no neurological disease. CXCL13 protein was localized in perivascular infiltrates and scattered infiltrating cells in lesion parenchyma. In the CSF of relapsing-remitting multiple sclerosis patients, both CXCL12 and CXCL13 were elevated. CXCL13, but not CXCL12, levels correlated strongly with intrathecal immunoglobulin production as well as the presence of B cells, plasma blasts and T cells. About 20% of CSF CD4+ cells and almost all B cells expressed the CXCL13 receptor CXCR5. In vitro, CXCL13 was produced by monocytes and at much higher levels by macrophages. CXCL13 mRNA and protein expression was induced by TNFalpha and IL-1beta but inhibited by IL-4 and IFNgamma. Together, CXCL12 and CXCL13 are elevated in active multiple sclerosis lesions and CXCL12 also in inactive lesions. The consequences of CXCL12 up-regulation could be manifold. CXCL12 localization on blood vessels indicates a possible role in leucocyte extravasation, and CXCL12 may contribute to plasma cell persistence since its receptor CXCR4 is retained during plasma cell differentiation. CXCL12 may contribute to axonal damage as it can become a neurotoxic mediator of cleavage by metalloproteases, which are present in multiple sclerosis lesions. The strong linkage of CXCL13 to immune cells and immunoglobulin levels in CSF suggests that this is one of the factors that attract and maintain B and T cells in inflamed CNS lesions. Therefore, both CXCL13 and CXCR5 may be promising therapeutic targets in multiple sclerosis.

Glatiramer acetate in multiple sclerosis: update on potential mechanisms of action.

Glatiramer acetate is a synthetic random copolymer approved for the immunomodulatory therapy of relapsing-type multiple sclerosis (MS). Previous work has focused on the effects of this drug on T cells, especially the glatiramer-acetate-induced shift of the cytokine profile towards those characteristic of T-helper-2 (Th2) cells. Glatiramer acetate was thought to bring about this Th2 shift by acting like an altered peptide ligand but more recent work has shown that the drug notably affects the properties of antigen-presenting cells, such as monocytes and dendritic cells. These new observations might offer an explanation for the previously observed Th2 shift. In this review, we focus on these new findings. We address several controversial issues, including the possible neurotrophic effects of glatiramer acetate, the potential role of neutralising antibodies to the drug, and attempts to develop biomarkers of the treatment response. Finally, we will think about how a better understanding of glatiramer acetate might help the development of new immunomodulatory agents for MS.

Preferential expression and function of Toll-like receptor 3 in human astrocytes.

In contrast to other tissues, the central nervous system (CNS) is essentially devoid of MHC expression and shielded from antibodies by the blood-brain barrier. Therefore, a rapid local innate immune response by resident brain cells is required to effectively fight infectious agents. This study analyzed the expression and function of Toll-like receptors (TLRs) in cultured human astrocytes. Quantitative PCR for TLRs 1 to 10 showed a basal expression of TLR3 that could be enhanced by IFN-gamma, IL-1beta, and IFN-beta. The other TLRs were barely detectable and not inducible by the same cytokines. IFN-gamma-activated astrocytes responded to TLR3 ligand poly (I:C) engagement with IL-6 production, while ligands of other TLRs, like LPS, lipoteichoic acid, peptidoglycan, flagellin, and CpG, had no effect. Poly (I:C) also triggered astrocyte production of TNF-alpha and the chemokines CCL2/MCP-1, CCL5/RANTES, CCL20/MIP-3alpha, and CXCL10/IP-10. The adapter molecules MyD88 (full length and short isoform), TIRAP/Mal, and TICAM-1/TRIF, which are required for TLR signaling, were all expressed by astrocytes. Thus, resting and activated human astrocytes express preferentially TLR3 and, upon TLR3 engagement, produce IL-6 and chemokines active on T cells, B cells, monocytes, and dendritic cells. These data indicate that astrocytes function as sentinels for viral infections in the CNS.

Multiple sclerosis: glatiramer acetate inhibits monocyte reactivity in vitro and in vivo.

It is widely assumed that glatiramer acetate (GA), an approved agent for the immunomodulatory treatment of multiple sclerosis, acts primarily as an antigen for T lymphocytes. Recent studies, however, indicated that in vitro, GA directly inhibits dendritic cells, a rare but potent type of professional antigen-presenting cell (APC). To investigate whether these in vitro observations are relevant to the actions of GA in vivo, we studied the effects of GA on monocytes, the major type of circulating APC. In a first series of experiments, we investigated the effects of GA on monocyte reactivity in vitro. Monocytes were stimulated with ligands for Toll-like receptor (TLR)-2 (peptidoglycan and lipoteichoic acid), TLR-4 [lipopolysaccharide (LPS)] and TLR-5 (flagellin), as well as two proinflammatory cytokines (interferon-gamma and granulocyte-monocyte colony-stimulating factor). Monocyte activation was measured by induction of the surface markers signalling lymphocytic activation molecule (SLAM), CD25 and CD69 (detected by cytofluorometry), and by production of monocyte-derived tumour necrosis factor (TNF)-alpha (detected by enzyme-linked immunospot assay). GA had a broad inhibitory effect on all measures of monocyte reactivity, regardless of which stimulator was used. It is unlikely that this reflects a simple toxic effect, because monocyte viability and CD14 expression were unaffected. In a second series of experiments, we investigated the properties of monocytes cultured ex vivo from eight GA-treated multiple sclerosis patients, eight untreated multiple sclerosis patients and eight healthy subjects. We found that LPS-induced SLAM expression and TNF-alpha production were significantly reduced in monocytes from GA-treated patients compared with controls. These results demonstrate for the first time that GA inhibits monocyte reactivity in vitro and in vivo, significantly extending the current concept of the mechanism of action of GA.

Immunological assay for assessing the efficacy of glatiramer acetate (Copaxone) in multiple sclerosis. A pilot study.

Recently we described an enzyme-linked immunoadsorbent spot (ELISPOT) assay allowing us to define an immunological response profile observed in multiple sclerosis patients treated with Copaxone (glatiramer acetate; GA) but not untreated subjects [4]. The profile encompasses three criteria, a) reduced proliferative response to GA (as observed with a standard primary proliferation assay); b) strong in vitro activation of interferon-gamma-producing T cells at high concentrations of GA (as detected by interferon-gamma ELISPOT); and c) activation of interleukin-4-producing T cells over a wider range of concentrations of GA (as detected by interleukin-4 ELISPOT). It is at present unknown whether the immunological response to GA correlates with the clinical response. To address this question we performed the pilot study reported here. We asked the major German multiple sclerosis centres to send us blood samples from all GA-treated patients who were going to discontinue treatment because of treatment failure. The clinical nonresponders either had an unchanged or increased exacerbation rate, or developed a secondary progressive course during GA treatment. Over more than one year, we prospectively collected 9 samples from clinical non-responders. We compared the immune response to GA of peripheral blood mononuclear cells from the 9 clinical nonresponders with 15 clinical responders, using a standard proliferation assay combined with ELISPOT assays for detection of interferon-gamma and interleukin-4 secreting cells. Thirteen (86 %) of the 15 clinical responders met at least 2 of the immunological response criteria mentioned above. In contrast, only 2 (22 %) of the 9 clinical nonresponders met two of the immunological criteria (p = 0.0006). We conclude that the ELISPOT assay may provide a promising additional tool for monitoring the treatment response in multiple sclerosis patients treated with GA.