Elisa Piscianz

Regulatory T-Cell Function Is Impaired in Celiac Disease

Celiac disease (CD) is characterized by intolerance to gluten and high risk of developing autoimmune phenomena. Possible defects in immune tolerance could have a role in the pathogenesis of the disease. As regulatory T-cells (Tregs) are the main population involved in maintaining peripheral tolerance, we investigated the number of these cells in celiac patients as compared with healthy donors. Moreover, we analyzed the suppressive function of CD4+CD25+ T-cells from celiac disease patients and controls on autologous responder T-cells (CD4+CD25−). The percentage of CD4+CD25+FOXP3+ cells was not different in celiacs and in healthy controls, and among positive cells the level of expression of the two regulatory markers was comparable. However, the suppressor activity of Tregs was significantly impaired in CD patients. These results suggest that a defect in Tregs function could play a role in the pathogenesis of CD and in CD-associated autoimmunity.

The immunosuppressive effect of Wharton's jelly stromal cells depends on the timing of their licensing and on lymphocyte activation

BACKGROUND Mesenchymal stromal cells (MSC) have been proven to have potent immunosuppressive action and hence have been proposed for the treatment of severe Graft Versus Host Disease. However, in most models, MSC were added at the same time of lymphocyte stimulation, which is quite different from what occurs in vivo. AIMS To investigate how the timing of lymphocyte activation and the exposure to activation-related cytokines (licensing) can influence the immunosuppressive action of Whartons jelly stromal cells (WJSC). METHODS WJSC, licensed or not with activation-related cytokines, were added lymphocytes the same time or 24 hours after their stimulation with phytohaemoagglutinin. Proliferation of lymphocytes and cytokines production was measured after three days co-culture. RESULTS Lymphocytes stimulated in the presence of WJSC displayed a dramatic decrease in proliferation and production of cytokines, in spite of normal expression of activation markers. The suppression was weakened when targeted lymphocytes were seperated by a membrane and partially rescued by the addition of exogenous l-tryptophan, suggesting a major role for indoleamine 2,3-dioxigenase with a probable paracrine effect. Licensing of WJSC increased the immunosuppressive effect, in both contact and non-contact settings. The timing of WJSC licensing was crucial for the immunosuppressive action. Lymphocytes pre-stimulated alone for 24 h, and added afterwards to non-licensed WJSC, showed normal or even increased proliferation. On the other hand, their proliferation was strongly inhibited by licensed WJSC. CONCLUSIONS WJSC have a potent immunosuppressive function best realized with direct contact, and increased by licensing signals before and during lymphocyte stimulation. Our results could contribute to the set up of new WJSC-based therapies for severe autoimmuno disorders.

Lovastatin-induced apoptosis is modulated by geranylgeraniol in a neuroblastoma cell line.

Mevalonic aciduria (MA), the most severe form of mevalonate kinase deficiency (MKD), is still an orphan drug disease and the pathogenetic mechanisms underlying neuronal dysfunction is still poorly understood. In our study we have investigated the apoptotic mechanism mediated by the exposure of the cultured neuroblastoma cell line, SH‐SY5Y, to lovastatin in absence or in presence of the isoprenoid, geranylgeraniol, with the aim of unraveling the pathogenesis of MA. Lovastatin, blocks the mevalonate pathway inhibiting the 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMG‐CR), an enzyme of the mevalonate pathway upstream the mevalonate kinase enzyme, reproducing biochemical features similar to those found in MKD.

Lovastatin induces apoptosis through the mitochondrial pathway in an undifferentiated SH-SY5Y neuroblastoma cell line

Lovastatin induces apoptosis through the mitochondrial pathway in an undifferentiated SH-SY5Y neuroblastoma cell line

Mevalonate Kinase Deficiency and Neuroinflammation: Balance between Apoptosis and Pyroptosis

Mevalonic aciduria, a rare autosomal recessive disease, represents the most severe form of the periodic fever, known as Mevalonate Kinase Deficiency. This disease is caused by the mutation of the MVK gene, which codes for the enzyme mevalonate kinase, along the cholesterol pathway. Mevalonic aciduria patients show recurrent fever episodes with associated inflammatory symptoms, severe neurologic impairments, or death, in early childhood. The typical neurodegeneration occurring in mevalonic aciduria is linked both to the intrinsic apoptosis pathway (caspase-3 and -9), which is triggered by mitochondrial damage, and to pyroptosis (caspase-1). These cell death mechanisms seem to be also related to the assembly of the inflammasome, which may, in turn, activate pro-inflammatory cytokines and chemokines. Thus, this particular molecular platform may play a crucial role in neuroinflammation mechanisms. Nowadays, a specific therapy is still lacking and the pathogenic mechanisms involving neuroinflammation and neuronal dysfunction have not yet been completely understood, making mevalonic aciduria an orphan drug disease. This review aims to analyze the relationship among neuroinflammation, mitochondrial damage, programmed cell death, and neurodegeneration. Targeting inflammation and degeneration in the central nervous system might help identify promising treatment approaches for mevalonic aciduria or other diseases in which these mechanisms are involved.

Long noncoding RNA GAS5: a novel marker involved in glucocorticoid response.

Glucocorticoids (GCs) exert their effects through regulation of gene expression after activation in the cytoplasm of the glucocorticoid receptor (GR) encoded by NR3C1 gene. A negative feedback mechanism resulting in GR autoregulation has been demonstrated through the binding of the activated receptor to intragenic sequences called GRE-like elements, contained in GR gene. The long noncoding RNA growth arrest-specific transcript 5 (GAS5) interacts with the activated GR suppressing its transcriptional activity. The aim of this study was to evaluate the possible role of GAS5 and NR3C1 gene expression in the antiproliferative effect of methylprednisolone in peripheral blood mononuclear cells and to correlate the expression with individual sensitivity to GCs. Subjects being poor responders to GCs presented higher levels of GAS5 and NR3C1 in comparison with good responders. We suggest that abnormal levels of GAS5 may alter GC effectiveness, probably interfering with the mechanism of GR autoregulation.

Knockdown of MVK does not lead to changes in NALP3 expression or activation

BackgroundMutations in the Mevalonate Kinase gene (MVK) are causes of a rare autoinflammatory disease: Mevalonate Kinase Deficiency and its more acute manifestation, Mevalonic Aciduria. The latter is characterized, among other features, by neuroinflammation, developmental delay and ataxia, due to failed cerebellar development or neuronal death through chronic inflammation. Pathogenesis of neuroinflammation in Mevalonate Kinase Deficiency and Mevalonic Aciduria has not yet been completely clarified, however different research groups have been suggesting the inflammasome complex as the key factor in the disease development. A strategy to mimic this disease is blocking the mevalonate pathway, using HMG-CoA reductase inhibitors (Statins), while knock-out mice for Mevalonate Kinase are non-vital and their hemyzygous (i.e only one copy of gene preserved) littermate display almost no pathological features.FindingsWe sought to generate a murine cellular model closely resembling the pathogenic conditions found in vivo, by direct silencing of Mevalonate Kinase gene. Knockdown of Mevalonate Kinase in a murine microglial cellular model (BV-2 cells) results in neither augmented NALP3 expression nor increase of apoptosis. On the contrary, statin treatment of BV-2 cells produces an increase both in Mevalonate Kinase and NALP3 expression.ConclusionsMKD deficiency could be due or affected by protein accumulation leading to NALP3 activation, opening novel questions about strategies to tackle this disease.

Inhibition of mesenchymal stromal cells by pre-activated lymphocytes and their culture media

IntroductionDespite having a proven immunosuppressive potential in vitro, human mesenchymal stromal cells (MSCs) are reported to display variable efficacy in vivo and, in fact, their proven benefit in the clinical practice is still limited and controversial.MethodsThe interplay between clinical grade MSCs and pre-activated donor lymphocytes or selected lymphocyte subsets was studied in vitro. The kinetics of MSC growth and viability was evaluated by adhesion-dependent changes of culture plate impedance and biochemically by a colorimetric assay. Activation of natural killer (NK) cells was assessed as well, using a flow cytometry assay.ResultsA strong inhibition of MSC growth was rapidly induced by the addition of pre-activated lymphocytes but not of resting lymphocytes. Inhibition seems not to be attributable to a single cell population, as similar results can be obtained by depleting NK cells or by using either selected CD4+ or CD8+ lymphocytes. In addition, conditioned medium (CM) from activated lymphocytes was able to inhibit MSC growth in a dose-dependent manner. Furthermore, licensing with IFN-γ partially protected MSCs from pre-activated lymphocytes but not from their CM. These results suggest an inhibitory role of lymphocyte-activation-derived substances. However, the identification of a single molecule responsible for MSC inhibition remained elusive, even if preliminary experiments showed that ATP and, to a lesser extent, TNF-α might play a role.ConclusionsThese results suggest that survival of MSCs can be affected by soluble mediators released by activated lymphocytes. Thus it can be hypothesized that MSC immunosuppressive action in vivo could be impaired by ongoing immune activation through the release of inflammatory mediators.

Lovastatin Dose-Dependently Potentiates the Pro-inflammatory Activity of Lipopolysaccharide Both In Vitro and In Vivo

Since contradictory findings have been reported on potential effects of statins in modulating the inflammatory response, we have analysed the biological activity of lovastatin both in vitro using the Raw 264.7 murine macrophagic cell line and in vivo using BALB/c mice. When added to Raw 264.7 cells in combination with lipopolysaccharide, lovastatin significantly potentiated the release of interleukin-1β, interleukin-6 and interleukin-12 with respect to lipopolysaccharide alone and showed an additive effect on the release of nitric oxide. Similarly, when lovastatin was intraperitoneally administrated to BALB/c mice, it did not induce any pro-inflammatory effect when used alone, but it significantly potentiated the pro-inflammatory activity of lipopolysaccharide, in terms of number of intraperitoneal cells and serum levels of serum amyloid A, interleukin-1β, interleukin-6 and interleukin-12. A potential clinical implication of our study is that lovastatin might exert a pro-inflammatory activity in subjects affected by inflammatory processes, with clinically evident or subclinical infections.

T cells stimulated in vitro have a suppressive function but do not contain only regulatory T cells

The generation of regulatory T cells (Tregs) in vitro represents an attractive possibility to set up cellular therapies that could prevent and cure autoimmune disorders. Different methods have been proposed to generate Tregs in vitro and to evaluate their phenotype and function. Moreover, the overlap between generation of activated and regulatory cells could often be underestimated. We showed that in vitro treatment of CD4+ CD25– lymphocytes with different stimuli leads to a good expression of CD25 and forkhead box P3 (FoxP3) on most cells, but to a full Treg phenotype (including CD127 negativity) in only a minor percentage of cells, ranging from 17·38% of cells treated with phytohaemagglutinin (PHA) to 50·91% of cells treated with T cell receptor (TCR) stimulation in association with transforming growth factor (TGF)‐β. Some suppressive activity was demonstrated for T cells activated with all the different stimuli. However, while suppression mediated by TCR/TGF‐β treated T cells was associated with an inhibition of both interleukin (IL)‐2 and interferon (IFN)‐γ in the co‐culture supernatant, the suppression observed for PHA‐activated cells occurred in the presence of large amounts of these cytokines. In conclusion, also taking into account other recent publications, caution should be taken in interpretation of data in the field of regulatory T cells.