Elena Bonechi

Plant microRNAs as novel immunomodulatory agents

An increasing body of literature is addressing the immuno-modulating functions of miRNAs which include paracrine signaling via exosome-mediated intercellular miRNA. In view of the recent evidence of intake and bioavailability of dietary miRNAs in humans and animals we explored the immuno-modulating capacity of plant derived miRNAs. Here we show that transfection of synthetic miRNAs or native miRNA-enriched fractions obtained from a wide range of plant species and organs modifies dendritic cells ability to respond to inflammatory agents by limiting T cell proliferation and consequently dampening inflammation. This immuno-modulatory effect appears associated with binding of plant miRNA on TLR3 with ensuing impairment of TRIF signaling. Similarly, in vivo, plant small RNAs reduce the onset of severity of Experimental Autoimmune Encephalomyelities by limiting dendritic cell migration and dampening Th1 and Th17 responses in a Treg-independent manner. Our results indicate a potential for therapeutic use of plant miRNAs in the prevention of chronic-inflammation related diseases.

Histamine Regulates Actin Cytoskeleton in Human Toll-like Receptor 4-activated Monocyte-derived Dendritic Cells Tuning CD4+ T Lymphocyte Response.

Histamine, a major mediator in allergic diseases, differentially regulates the polarizing ability of dendritic cells after Toll-like receptor (TLR) stimulation, by not completely explained mechanisms. In this study we investigated the effects of histamine on innate immune reaction during the response of human monocyte-derived DCs (mDCs) to different TLR stimuli: LPS, specific for TLR4, and Pam3Cys, specific for heterodimer molecule TLR1/TLR2. We investigated actin remodeling induced by histamine together with mDCs phenotype, cytokine production, and the stimulatory and polarizing ability of Th0. By confocal microscopy and RT-PCR expression of Rac1/CdC42 Rho GTPases, responsible for actin remodeling, we show that histamine selectively modifies actin cytoskeleton organization induced by TLR4, but not TLR2 and this correlates with increased IL4 production and decreased IFNγ by primed T cells. We also demonstrate that histamine-induced cytoskeleton organization is at least in part mediated by down-regulation of small Rho GTPase CdC42 and the protein target PAK1, but not by down-regulation of Rac1. The presence and relative expression of histamine receptors HR1–4 and TLRs were determined as well. Independently of actin remodeling, histamine down-regulates IL12p70 and CXCL10 production in mDCs after TLR2 and TLR4 stimulation. We also observed a trend of IL10 up-regulation that, despite previous reports, did not reach statistical significance.

Human dendritic cell activation induced by a permannosylated dendron containing an antigenic GM3-lactone mimetic

Summary Vaccination strategies based on dendritic cells (DCs) armed with specific tumor antigens have been widely exploited due the properties of these immune cells in coordinating an innate and adaptive response. Here, we describe the convergent synthesis of the bifunctional multivalent glycodendron 5, which contains nine residues of mannose for DC targeting and one residue of an immunogenic mimetic of a carbohydrate melanoma associated antigen. The immunological assays demonstrated that the glycodendron 5 is able to induce human immature DC activation in terms of a phenotype expression of co-stimulatory molecules expression and MHCII. Furthermore, DCs activated by the glycodendron 5 stimulate T lymphocytes to proliferate in a mixed lymphocytes reaction (MLR).

Oleoylethanolamide treatment affects gut microbiota composition and the expression of intestinal cytokines in Peyer’s patches of mice

The lipid sensor oleoylethanolamide (OEA), an endogenous high-affinity agonist of peroxisome proliferator-activated receptor-α (PPAR-α) secreted in the proximal intestine, is endowed with several distinctive homeostatic properties, such as control of appetite, anti-inflammatory activity, stimulation of lipolysis and fatty acid oxidation. When administered exogenously, OEA has beneficial effects in several cognitive paradigms; therefore, in all respects, OEA can be considered a hormone of the gut-brain axis. Here we report an unexplored modulatory effect of OEA on the intestinal microbiota and on immune response. Our study shows for the first time that sub-chronic OEA administration to mice fed a normal chow pellet diet, changes the faecal microbiota profile, shifting the Firmicutes:Bacteroidetes ratio in favour of Bacteroidetes (in particular Bacteroides genus) and decreasing Firmicutes (Lactobacillus), and reduces intestinal cytokines expression by immune cells isolated from Peyer’s patches. Our results suggest that sub-chronic OEA treatment modulates gut microbiota composition towards a “lean-like phenotype”, and polarises gut-specific immune responses mimicking the effect of a diet low in fat and high in polysaccharides content.

Myelin-specific T cells carry and release magnetite PGLA-PEG COOH nanoparticles in the mouse central nervous system

Progress in nanotechnology has determined new strategies concerning drug delivery into the central nervous system for the treatment of degenerative and inflammatory diseases. To date, brain targeting through systemic drug administration, even in a nano-composition, is often unsuccessful. Therefore, we investigated the possibility of loading T lymphocytes with PGLA–PEG COOH magnetite nanoparticles (30 nm), which can be built up to easily bind drugs and monoclonal antibodies, and to exploit the ability of activated T cells to cross the blood–brain barrier and infiltrate the brain parenchyma. Iron oxide nanoparticles have been widely used in biomedical applications due to their theranostic properties and are therefore a well-established nanomaterial. The magnetite core is easily hybridized with polymeric compounds that may enhance the possibility of the nanoparticles entering cells with low phagocytic properties. Taking advantage of these material characteristics, after in vitro assessment of the viability and functionality of nano-loaded MOG35–55 specific T cells, we transferred cells containing the nano-cargo into naive mice affected by experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. By means of histological and immunohistological methods, we were able to identify the nano-loaded T cells in the central nervous system. Our data demonstrated that T cells containing nanomaterials hold the possibility of carrying and releasing nanoparticles in the brain.

Bridging pro-inflammatory signals, synaptic transmission and protection in spinal explants in vitro

Multiple sclerosis is characterized by tissue atrophy involving the brain and the spinal cord, where reactive inflammation contributes to the neurodegenerative processes. Recently, the presence of synapse alterations induced by the inflammatory responses was suggested by experimental and clinical observations, in experimental autoimmune encephalomyelitis mouse model and in patients, respectively. Further knowledge on the interplay between pro-inflammatory agents, neuroglia and synaptic dysfunction is crucial to the design of unconventional protective molecules. Here we report the effects, on spinal cord circuits, of a cytokine cocktail that partly mimics the signature of T lymphocytes sub population Th1. In embryonic mouse spinal organ-cultures, containing neuronal cells and neuroglia, cytokines induced inflammatory responses accompanied by a significant increase in spontaneous synaptic activity. We suggest that cytokines specifically altered signal integration in spinal networks by speeding the decay of GABAA responses. This hypothesis is supported by the finding that synapse protection by a non-peptidic NGF mimetic molecule prevented both the changes in the time course of GABA events and in network activity that were left unchanged by the cytokine production from astrocytes and microglia present in the cultured tissue. In conclusion, we developed an important tool for the study of synaptic alterations induced by inflammation, that takes into account the role of neuronal and not neuronal resident cells.

CSF/serum matrix metallopeptidase‐9 ratio discriminates neuro Behçet from multiple sclerosis

In neuro Behçet disease with multiple sclerosis‐like features, diagnosis could be challenging. Here, we studied the cerebrospinal fluid and serum inflammatory profile of 11 neuro Behçet and 21 relapsing‐remitting multiple sclerosis patients. Between the soluble factors analyzed (MMP9, TNFα, IL6, CXCL13, CXCL10, CXCL8, IFNγ, IL10, IL17, IL23, and others) we found MMP9 increased in neuro Behçet serum compared to multiple sclerosis and decreased in cerebrospinal fluid. Furthermore, neuro Behçet analysis of circulating natural killer CD56DIM subset suggests their potential involvement in increased MMP9 production. We believe that these findings may have a translational utility in clinical practice.

The Adaptor Protein Rai/ShcC Promotes Astrocyte-Dependent Inflammation during Experimental Autoimmune Encephalomyelitis

Th17 cells have been casually associated to the pathogenesis of autoimmune disease. We have previously demonstrated that Rai/ShcC, a member of the Shc family of adaptor proteins, negatively regulates Th17 cell differentiation and lupus autoimmunity. In this study, we have investigated the pathogenic outcome of the Th17 bias associated with Rai deficiency on multiple sclerosis development, using the experimental autoimmune encephalomyelitis (EAE) mouse model. We found that, unexpectedly, EAE was less severe in Rai−/− mice compared with their wild-type counterparts despite an enhanced generation of myelin-specific Th17 cells that infiltrated into the CNS. Nevertheless, when adoptively transferred into immunodeficient Rai+/+ mice, these cells promoted a more severe disease compared with wild-type encephalitogenic Th17 cells. This paradoxical phenotype was caused by a dampened inflammatory response of astrocytes, which were found to express Rai, to IL-17. The results provide evidence that Rai plays opposite roles in Th17 cell differentiation and astrocyte activation, with the latter dominant over the former in EAE, highlighting this adaptor as a potential novel target for the therapy of multiple sclerosis.

Increased CXCL10 expression in MS MSCs and monocytes is unaffected by AHSCT

To confirm CXCL10 over production in bone marrow mesenchymal stem cells (MSCs) and circulating monocytes isolated from multiple sclerosis patients (MS) and identify predate cell molecular signature; to extend this analysis after autologous hematopoietic stem cell transplantation (AHSCT) to test if therapy has modifying effects on MSCs and circulating monocytes.