Ermanna Turano

ADIPOSE-DERIVED MESENCHYMAL STEM CELLS AMELIORATE CHRONIC EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for neurological autoimmune diseases; previous studies have shown that treatment with bone marrow‐derived MSCs induces immune modulation and reduces disease severity in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Here we show that intravenous administration of adipose‐derived MSCs (ASCs) before disease onset significantly reduces the severity of EAE by immune modulation and decreases spinal cord inflammation and demyelination. ASCs preferentially home into lymphoid organs but also migrates inside the central nervous system (CNS). Most importantly, administration of ASCs in chronic established EAE significantly ameliorates the disease course and reduces both demyelination and axonal loss, and induces a Th2‐type cytokine shift in T cells. Interestingly, a relevant subset of ASCs expresses activated α4 integrins and adheres to inflamed brain venules in intravital microscopy experiments. Bioluminescence imaging shows that α4 integrins control ASC accumulation in inflamed CNS. Importantly, we found that ASC cultures produce basic fibroblast growth factor, brain‐derived growth factor, and platelet‐derived growth factor‐AB. Moreover, ASC infiltration within demyelinated areas is accompanied by increased number of endogenous oligodendrocyte progenitors. In conclusion, we show that ASCs have clear therapeutic potential by a bimodal mechanism, by suppressing the autoimmune response in early phases of disease as well as by inducing local neuroregeneration by endogenous progenitors in animals with established disease. Overall, our data suggest that ASCs represent a valuable tool for stem cell–based therapy in chronic inflammatory diseases of the CNS. STEM CELLS 2009;27:2624–2635

Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin

Inflammation is a pathological hallmark of Alzheimers disease, and innate immune cells have been shown to contribute to disease pathogenesis. In two transgenic models of Alzheimers disease (5xFAD and 3xTg-AD mice), neutrophils extravasated and were present in areas with amyloid-β (Aβ) deposits, where they released neutrophil extracellular traps (NETs) and IL-17. Aβ42 peptide triggered the LFA-1 integrin high-affinity state and rapid neutrophil adhesion to integrin ligands. In vivo, LFA-1 integrin controlled neutrophil extravasation into the CNS and intraparenchymal motility. In transgenic Alzheimers disease models, neutrophil depletion or inhibition of neutrophil trafficking via LFA-1 blockade reduced Alzheimers disease–like neuropathology and improved memory in mice already showing cognitive dysfunction. Temporary depletion of neutrophils for 1 month at early stages of disease led to sustained improvements in memory. Transgenic Alzheimers disease model mice lacking LFA-1 were protected from cognitive decline and had reduced gliosis. In humans with Alzheimers disease, neutrophils adhered to and spread inside brain venules and were present in the parenchyma, along with NETs. Our results demonstrate that neutrophils contribute to Alzheimers disease pathogenesis and cognitive impairment and suggest that the inhibition of neutrophil trafficking may be beneficial in Alzheimers disease.

Murine adipose-derived mesenchymal stromal cell vesicles: in vitro clues for neuroprotective and neuroregenerative approaches

BACKGROUND AIMS Adipose-derived mesenchymal stromal cells (ASC) are known to promote neuroprotection and neuroregeneration in vitro and in vivo. These biological effects are probably mediated by paracrine mechanisms. In recent years, nanovesicles (NV) and microvesicles (MV) have been shown to play a major role in cell-to-cell communication. We tested the efficacy of NV and MV obtained from ASC in mediating neuroprotection and neuroregeneration in vitro. METHODS We exposed neuronal cells (both cell line and primary cultures) to oxidative stress in the presence or not of NV or MV. RESULTS In this experimental setting, we found that low doses of NV or MV protected neurons from apoptotic cell death. We then assessed the neuroregenerative effect of NV/MV in cerebellar slice cultures demyelinated with lysophosphatidylcholine. We observed that low but not higher doses of NV and MV increased the process of remyelination and activated nestin-positive oligodendroglial precursors. CONCLUSIONS Taken together, our data in vitro support the relevance of ASC vesicles as a source of protecting and regenerating factors that might modulate the microenvironment in neuro-inflammatory as well as in neurodegenerative disorders. The present findings may suggest that stromal cell-derived vesicles might represent a potential therapeutic tool, enabling the safe administration of stromal cell effector factors, avoiding the cellular counterpart.

2D immunomic approach for the study of IgG autoantibodies in the experimental model of multiple sclerosis.

2D-immunomics may be useful in the identification of autoantigens in neurological autoimmune diseases, but its application may be limited by denaturation of target proteins. Here we compared the capacity of a single or multiple antigens to elicit autoantibodies targeting multiple neural autoantigens by ELISA and 2D-immunomics. We induced experimental autoimmune encephalomyelitis (EAE) with MBP peptide(89-104), total MBP or spinal cord homogenate. Both techniques showed anti-MBP IgG only after immunization with total MBP. In addition, 2D-immunomics revealed the presence in EAE mice of autoantibodies targeting other neural proteins, some displaying partial sequence homology with MBP. The present finding by 2D-immunomics of multiple neural proteins targeted by autoantibodies generated by a single antigen may help to explain the complex autoimmune response observed in multiple sclerosis.

Nanovesicles from adipose-derived mesenchymal stem cells inhibit T lymphocyte trafficking and ameliorate chronic experimental autoimmune encephalomyelitis

Cell based-therapies represent promising strategies for the treatment of neurological diseases. We have previously shown that adipose stem cells (ASC) ameliorate chronic experimental autoimmune encephalomyelitis (EAE). Recent evidence indicates that most ASC paracrine effects are mediated by extracellular vesicles, i.e. micro- and nanovesicles (MVs and NVs). We show that preventive intravenous administration of NVs isolated from ASC (ASC-NVs) before disease onset significantly reduces the severity of EAE and decreases spinal cord inflammation and demyelination, whereas therapeutic treatment with ASC-NVs does not ameliorate established EAE. This treatment marginally inhibits antigen-specific T cell activation, while reducing microglial activation and demyelination in the spinal cord. Importantly, ASC-NVs inhibited integrin-dependent adhesion of encephalitogenic T cells in vitro, with no effect on adhesion molecule expression. In addition, intravital microscopy showed that encephalitogenic T cells treated with ASC NVs display a significantly reduced rolling and firm adhesion in inflamed spinal cord vessels compared to untreated cells. Our results show that ASC-NVs ameliorate EAE pathogenesis mainly by inhibiting T cell extravasation in the inflamed CNS, suggesting that NVs may represent a novel therapeutic approach in neuro-inflammatory diseases, enabling the safe administration of ASC effector factors.

Defining an immune signature predictive of glioma progression

astrocytic end feet that form, togetherwith the endothelial cells, pericytes and the basal lamina, the blood brain barrier (BBB). The close interaction of these components, which is necessary for maintaining the integrity of the BBB, is still poorly understood. In this study we investigated the time course and structural correlates of BBB-damage in a model of NMO and the impact of astrocyte loss on the breakdown and restoration of the BBB. Using an in vivo model of focal NMO in rats based on injection of a recombinant human anti-AQP4 antibody and complement, we found a prominent opening of the BBB to endogenous albumin, immunoglobulin G and fibrinogen and the exogenousmarkermolecules FITC-albumin and Texas Red Cadaverine at early stages (6 h) of lesion development. The extravasation of these molecules was on protein level accompanied by the loss of the tight junction (TJ)molecule occludin from the blood vessels in the lesions, while the TJ molecules claudin5 and claudin3 were largely preserved. On mRNA level a compensatory upregulation of these TJ molecules was observed at the same time. Interestingly, the BBB integrity was rapidly restored (within 24 h), although astrocyte loss was highest at this time point and occludin was still lacking from the lesion, thus indicating compensatory mechanisms at the BBB independent of astrocyte signaling. In conclusion, there is a prominent breakdown of the BBB in early stages of experimental NMO associated with loss of astrocytes and tight junction molecules. Several hours later, the BBB is restored again, even though astrocytes are still absent from the lesion.

Role of nanovesicles from macrophages/microglia in the cross-talk between glioma cells and microenvironment

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. GBM is characterized by cellular heterogeneity, rapid proliferation, angiogenesis and extensive invasion. Glioma tumor cells actively recruit to the tumor site microglia as well as peripheral macrophages, named Tumor Associate Macrophages (TAMs). TAMs have been shown to be deeply involved in tumor microenvironment by their ability to induce immunosuppression, angiogenesis and invasion. Macrophages that infiltrate cancer tissues can be classified inM1 (proinflammatory) and M2 (anti-inflammatory) according to their “activation” state. M1 macrophages produce type I proinflammatory cytokines, participate in antigen presentation and have an anti-tumorigenic role. Conversely, M2 macrophages produce type II cytokines, promote antiinflammatory responses and have pro-tumorigenic functions. Recent antitumor strategies are aiming to target TAMs with different approaches: inhibiting their recruitment, suppressing their survival, enhancing M1 like and blocking M2 activities. We focused our attention on M1 polarized macrophages and how they could influence glioma cells. We attempted to explore whether soluble factors secreted by M1 polarized microglia/macrophages could impact the cell fate of U251 glioma cells. Our preliminary experiments showed that M1 conditioned medium (M1CM) inhibits tumor cell proliferation aswell as promotes apoptosis. Extracellular vesicles have emerged as important mediators of intercellular communication in cancer. Among them, exosomes are defined as vesicles characterized by a size of 30–100 nm in diameter and microvesicles from 50 nm to 1000 nm, both recognized as important mediators of cell-to-cell communication. Currently studies in other type of cancers indicate that nanovesicles present in the CMplay a role in the modulation of tumor microenvironment. In line with these observations, we found that treatment of U251 cells with exosomes derived from M1 polarized microglia/macrophages decreases glioma cell proliferation. Interestingly, both M1 exosomes and microvesicles were more effective thanM1 total conditionedmedium.Wehypothesize that exosome re-polarization toward an M1 phenotype might oppose glioma progression. These findings shed new light on the complex communication networks in theGBMmicroenvironment and open new future therapeutic strategies.

NEUTROPHILS INDUCE ALZHEIMER'S-LIKE DISEASE VIA LFA-1-INTEGRIN AND NEUTROPHIL EXTRACELLULAR TRAPS

Methods: Confocal microscopy studies were performed to evaluate inflammation mechanisms. Intra-vital microscopy studies using two-photon microscopy were performed to visualize and analyze the movement of neutrophils inside brain vessels and in the parenchyma. In vitro rapid adhesion assays were performed on integrin ligands whereas and integrin affinity was measured using Image Stream technology. Neuropathological studies, fear conditioning and Y maze tests were performed to analyze the effect of inflammation mechanism inhibition on disease.