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Dive into the research topics where Michela Riba is active.

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Featured researches published by Michela Riba.


Blood | 2013

A novel function for FOXP3 in humans: intrinsic regulation of conventional T cells

Alicia N. McMurchy; Jana Gillies; Maria Concetta Gizzi; Michela Riba; Jose Manuel Garcia-Manteiga; Davide Cittaro; Dejan Lazarevic; Sara Di Nunzio; Ignazio Stefano Piras; Alessandro Bulfone; Maria Grazia Roncarolo; Elia Stupka; Rosa Bacchetta; Megan K. Levings

The role of forkhead box P3 (FOXP3) is well-established in T-regulatory cells, but the function of transient FOXP3 expression in activated human conventional T (Tconv) cells is unknown. In the present study, we used 2 approaches to determine the role of FOXP3 in human Tconv cells. First, we obtained Tconv clones from a female subject who is hemizygous for a null mutation in FOXP3, allowing the comparison of autologous T-cell clones that do or do not express FOXP3. Second, we knocked down activation-induced FOXP3 in Tconv cells from healthy donors with small interfering RNAagainst FOXP3. We found that FOXP3-deficient Tconv cells proliferate more and produce more cytokines than wild-type Tconv cells and have differential expression of 274 genes. We also investigated the role of FOXP3 in Th1 and Th17 cells and found that the expression of activation-induced FOXP3 was higher and more sustained in Th17 cells compared with Th1 cells. Knocking down FOXP3 expression in Th17 cells significantly increased the production of IFN-γ and decreased the expression of CCR4, but had no effect on IL-17 expression. These data reveal a novel function of FOXP3 in Tconv cells and suggest that expression of this protein is important in the function of multiple CD4(+) T-cell lineages.


Proceedings of the National Academy of Sciences of the United States of America | 2012

Amino acid starvation induces reactivation of silenced transgenes and latent HIV-1 provirus via down-regulation of histone deacetylase 4 (HDAC4)

Ilaria Palmisano; Giulia Della Chiara; Rosa Lucia D’Ambrosio; Claudia Huichalaf; Paola Brambilla; Silvia Corbetta; Michela Riba; Rosanna Piccirillo; Sergio Valente; Giorgio Casari; Antonello Mai; Filippo Martinelli Boneschi; Davide Gabellini; Guido Poli; Maria Vittoria Schiaffino

The epigenetic silencing of exogenous transcriptional units integrated into the genome represents a critical problem both for long-term gene therapy efficacy and for the eradication of latent viral infections. We report here that limitation of essential amino acids, such as methionine and cysteine, causes selective up-regulation of exogenous transgene expression in mammalian cells. Prolonged amino acid deprivation led to significant and reversible increase in the expression levels of stably integrated transgenes transcribed by means of viral or human promoters in HeLa cells. This phenomenon was mediated by epigenetic chromatin modifications, because histone deacetylase (HDAC) inhibitors reproduced starvation-induced transgene up-regulation, and transcriptome analysis, ChIP, and pharmacological and RNAi approaches revealed that a specific class II HDAC, namely HDAC4, plays a critical role in maintaining the silencing of exogenous transgenes. This mechanism was also operational in cells chronically infected with HIV-1, the etiological agent of AIDS, in a latency state. Indeed, both amino acid starvation and pharmacological inhibition of HDAC4 promoted reactivation of HIV-1 transcription and reverse transcriptase activity production in HDAC4+ ACH-2 T-lymphocytic cells but not in HDAC4− U1 promonocytic cells. Thus, amino acid deprivation leads to transcriptional derepression of silenced transgenes, including integrated plasmids and retroviruses, by a process involving inactivation or down-regulation of HDAC4. These findings suggest that selective targeting of HDAC4 might represent a unique strategy for modulating the expression of therapeutic viral vectors, as well as that of integrated HIV-1 proviruses in latent reservoirs without significant cytotoxicity.


PLOS ONE | 2013

A Strong Anti-Inflammatory Signature Revealed by Liver Transcription Profiling of Tmprss6−/− Mice

Michela Riba; Marco Rausa; Melissa Sorosina; Davide Cittaro; Jose Manuel Garcia Manteiga; Antonella Nai; Alessia Pagani; Filippo Martinelli-Boneschi; Elia Stupka; Clara Camaschella; Laura Silvestri

Control of systemic iron homeostasis is interconnected with the inflammatory response through the key iron regulator, the antimicrobial peptide hepcidin. We have previously shown that mice with iron deficiency anemia (IDA)-low hepcidin show a pro-inflammatory response that is blunted in iron deficient-high hepcidin Tmprss6 KO mice. The transcriptional response associated with chronic hepcidin overexpression due to genetic inactivation of Tmprss6 is unknown. By using whole genome transcription profiling of the liver and analysis of spleen immune-related genes we identified several functional pathways differentially expressed in Tmprss6 KO mice, compared to IDA animals and thus irrespective of the iron status. In the effort of defining genes potentially targets of Tmprss6 we analyzed liver gene expression changes according to the genotype and independently of treatment. Tmprss6 inactivation causes down-regulation of liver pathways connected to immune and inflammatory response as well as spleen genes related to macrophage activation and inflammatory cytokines production. The anti-inflammatory status of Tmprss6 KO animals was confirmed by the down-regulation of pathways related to immunity, stress response and intracellular signaling in both liver and spleen after LPS treatment. Opposite to Tmprss6 KO mice, Hfe−/− mice are characterized by iron overload with inappropriately low hepcidin levels. Liver expression profiling of Hfe−/− deficient versus iron loaded mice show the opposite expression of some of the genes modulated by the loss of Tmprss6. Altogether our results confirm the anti-inflammatory status of Tmprss6 KO mice and identify new potential target pathways/genes of Tmprss6.


Proceedings of the National Academy of Sciences of the United States of America | 2016

miR-17∼92 family clusters control iNKT cell ontogenesis via modulation of TGF-β signaling

Maya Fedeli; Michela Riba; Jose Manuel Garcia Manteiga; Lei Tian; Valentina Viganò; Grazisa Rossetti; Massimiliano Pagani; Changchun Xiao; Adrian Liston; Elia Stupka; Davide Cittaro; Sergio Abrignani; Paolo Provero; Paolo Dellabona; Giulia Casorati

Significance CD1d-restricted invariant natural killer T (iNKT) cells are innate-like T lymphocytes that play fundamental roles in cancer, autoimmunity, and infections. iNKT cells acquire effector functions already in the thymus, because of a distinct developmentally regulated genetic program that is critically controlled by miRNAs. Our study unveils the unexpected requirement for miRNA-dependent fine-tuning of TGF-β signaling in the control of iNKT cell development and functional differentiation. The targeting of a lineage-specific cytokine signaling by miRNA represents a previously unknown level of developmental regulation in the thymus. Furthermore, our study provides a comprehensive atlas of miRNA-regulated molecular pathways involved in iNKT cell ontogenesis, and highlights molecular pathways targeted by defined miRNAs that are predicted to be involved in the development and maturation of CD1d-restricted iNKT cells. Invariant natural killer T cells (iNKT) cells are T lymphocytes displaying innate effector functions, acquired through a distinct thymic developmental program regulated by microRNAs (miRNAs). Deleting miRNAs by Dicer ablation (Dicer KO) in thymocytes selectively impairs iNKT cell survival and functional differentiation. To unravel this miRNA-dependent program, we systemically identified transcripts that were differentially expressed between WT and Dicer KO iNKT cells at different differentiation stages and predicted to be targeted by the iNKT cell-specific miRNAs. TGF-β receptor II (TGF-βRII), critically implicated in iNKT cell differentiation, was found up-regulated in iNKT Dicer KO cells together with enhanced TGF-β signaling. miRNA members of the miR-17∼92 family clusters were predicted to target Tgfbr2 mRNA upon iNKT cell development. iNKT cells lacking all three miR-17∼92 family clusters (miR-17∼92, miR-106a∼363, miR-106b∼25) phenocopied both increased TGF-βRII expression and signaling, and defective effector differentiation, displayed by iNKT Dicer KO cells. Consistently, genetic ablation of TGF-β signaling in the absence of miRNAs rescued iNKT cell differentiation. These results elucidate the global impact of miRNAs on the iNKT cell developmental program and uncover the targeting of a lineage-specific cytokine signaling by miRNAs as a mechanism regulating innate-like T-cell development and effector differentiation.


PLOS ONE | 2014

Genome Wide Identification of Aberrant Alternative Splicing Events in Myotonic Dystrophy Type 2

Alessandra Perfetti; Simona Greco; Pasquale Fasanaro; Enrico Bugiardini; Rosanna Cardani; Jose Manuel Garcia Manteiga; Michela Riba; Davide Cittaro; Elia Stupka; Giovanni Meola; Fabio Martelli

Myotonic dystrophy type 2 (DM2) is a genetic, autosomal dominant disease due to expansion of tetraplet (CCTG) repetitions in the first intron of the ZNF9/CNBP gene. DM2 is a multisystemic disorder affecting the skeletal muscle, the heart, the eye and the endocrine system. According to the proposed pathological mechanism, the expanded tetraplets have an RNA toxic effect, disrupting the splicing of many mRNAs. Thus, the identification of aberrantly spliced transcripts is instrumental for our understanding of the molecular mechanisms underpinning the disease. The aim of this study was the identification of new aberrant alternative splicing events in DM2 patients. By genome wide analysis of 10 DM2 patients and 10 controls (CTR), we identified 273 alternative spliced exons in 218 genes. While many aberrant splicing events were already identified in the past, most were new. A subset of these events was validated by qPCR assays in 19 DM2 and 15 CTR subjects. To gain insight into the molecular pathways involving the identified aberrantly spliced genes, we performed a bioinformatics analysis with Ingenuity system. This analysis indicated a deregulation of development, cell survival, metabolism, calcium signaling and contractility. In conclusion, our genome wide analysis provided a database of aberrant splicing events in the skeletal muscle of DM2 patients. The affected genes are involved in numerous pathways and networks important for muscle physio-pathology, suggesting that the identified variants may contribute to DM2 pathogenesis.


Cancer Research | 2017

T Cells Redirected to a Minor Histocompatibility Antigen Instruct Intratumoral TNFα Expression and Empower Adoptive Cell Therapy for Solid Tumors

Teresa Manzo; Tabea Sturmheit; Veronica Basso; Elisabetta Petrozziello; Rodrigo Hess Michelini; Michela Riba; Massimo Freschi; Angela Rita Elia; Matteo Grioni; Flavio Curnis; Maria Pia Protti; Ton N. M. Schumacher; Reno Debets; Melody A. Swartz; Angelo Corti; Matteo Bellone; Anna Mondino

Donor-derived allogeneic T cells evoke potent graft versus tumor (GVT) effects likely due to the simultaneous recognition of tumor-specific and host-restricted minor histocompatibility (H) antigens. Here we investigated whether such effects could be reproduced in autologous settings by TCR gene-engineered lymphocytes. We report that T cells redirected either to a broadly expressed Y-encoded minor H antigen or to a tumor-associated antigen, although poorly effective if individually transferred, when simultaneously administered enabled acute autochthonous tumor debulking and resulted in durable clinical remission. Y-redirected T cells proved hyporesponsive in peripheral lymphoid organs, whereas they retained effector function at the tumor site, where in synergy with tumor-redirected lymphocytes, they instructed TNFα expression, endothelial cell activation, and intratumoral T-cell infiltration. While neutralizing TNFα hindered GVT effects by the combined T-cell infusion, a single injection of picogram amounts of NGR-TNF, a tumor vessel-targeted TNFα derivative currently in phase III clinical trials, substituted for Y-redirected cells and enabled tumor debulking by tumor-redirected lymphocytes. Together, our results provide new mechanistic insights into allogeneic GVT, validate the importance of targeting the tumor and its associated stroma, and prove the potency of a novel combined approach suitable for immediate clinical implementation. Cancer Res; 77(3); 658-71. ©2016 AACR.


Scientific Reports | 2016

Revealing the acute asthma ignorome: characterization and validation of uninvestigated gene networks.

Michela Riba; Jose Manuel Garcia Manteiga; Berislav Bošnjak; Davide Cittaro; Pavol Mikolka; Connie Le; Michelle M. Epstein; Elia Stupka

Systems biology provides opportunities to fully understand the genes and pathways in disease pathogenesis. We used literature knowledge and unbiased multiple data meta-analysis paradigms to analyze microarray datasets across different mouse strains and acute allergic asthma models. Our combined gene-driven and pathway-driven strategies generated a stringent signature list totaling 933 genes with 41% (440) asthma-annotated genes and 59% (493) ignorome genes, not previously associated with asthma. Within the list, we identified inflammation, circadian rhythm, lung-specific insult response, stem cell proliferation domains, hubs, peripheral genes, and super-connectors that link the biological domains (Il6, Il1ß, Cd4, Cd44, Stat1, Traf6, Rela, Cadm1, Nr3c1, Prkcd, Vwf, Erbb2). In conclusion, this novel bioinformatics approach will be a powerful strategy for clinical and across species data analysis that allows for the validation of experimental models and might lead to the discovery of novel mechanistic insights in asthma.


Embo Molecular Medicine | 2017

Lentiviral vectors escape innate sensing but trigger p53 in human hematopoietic stem and progenitor cells

Francesco Piras; Michela Riba; Carolina Petrillo; Dejan Lazarevic; Ivan Cuccovillo; Sara Bartolaccini; Elia Stupka; Bernhard Gentner; Davide Cittaro; Luigi Naldini; Anna Kajaste-Rudnitski

Clinical application of lentiviral vector (LV)‐based hematopoietic stem and progenitor cells (HSPC) gene therapy is rapidly becoming a reality. Nevertheless, LV‐mediated signaling and its potential functional consequences on HSPC biology remain poorly understood. We unravel here a remarkably limited impact of LV on the HSPC transcriptional landscape. LV escaped innate immune sensing that instead led to robust IFN responses upon transduction with a gamma‐retroviral vector. However, reverse‐transcribed LV DNA did trigger p53 signaling, activated also by non‐integrating Adeno‐associated vector, ultimately leading to lower cell recovery ex vivo and engraftment in vivo. These effects were more pronounced in the short‐term repopulating cells while long‐term HSC frequencies remained unaffected. Blocking LV‐induced signaling partially rescued both apoptosis and engraftment, highlighting a novel strategy to further dampen the impact of ex vivo gene transfer on HSPC. Overall, our results shed light on viral vector sensing in HSPC and provide critical insight for the development of more stealth gene therapy strategies.


PLOS ONE | 2018

Amino acid deprivation triggers a novel GCN2-independent response leading to the transcriptional reactivation of non-native DNA sequences

Annarosaria De Vito; Massimo Lazzaro; Ilaria Palmisano; Davide Cittaro; Michela Riba; Dejan Lazarevic; Makoto Bannai; Davide Gabellini; Maria Vittoria Schiaffino

In a variety of species, reduced food intake, and in particular protein or amino acid (AA) restriction, extends lifespan and healthspan. However, the underlying epigenetic and/or transcriptional mechanisms are largely unknown, and dissection of specific pathways in cultured cells may contribute to filling this gap. We have previously shown that, in mammalian cells, deprivation of essential AAs (methionine/cysteine or tyrosine) leads to the transcriptional reactivation of integrated silenced transgenes, including plasmid and retroviral vectors and latent HIV-1 provirus, by a process involving epigenetic chromatic remodeling and histone acetylation. Here we show that the deprivation of methionine/cysteine also leads to the transcriptional upregulation of endogenous retroviruses, suggesting that essential AA starvation affects the expression not only of exogenous non-native DNA sequences, but also of endogenous anciently-integrated and silenced parasitic elements of the genome. Moreover, we show that the transgene reactivation response is highly conserved in different mammalian cell types, and it is reproducible with deprivation of most essential AAs. The General Control Non-derepressible 2 (GCN2) kinase and the downstream integrated stress response represent the best candidates mediating this process; however, by pharmacological approaches, RNA interference and genomic editing, we demonstrate that they are not implicated. Instead, the response requires MEK/ERK and/or JNK activity and is reproduced by ribosomal inhibitors, suggesting that it is triggered by a novel nutrient-sensing and signaling pathway, initiated by translational block at the ribosome, and independent of mTOR and GCN2. Overall, these findings point to a general transcriptional response to essential AA deprivation, which affects the expression of non-native genomic sequences, with relevant implications for the epigenetic/transcriptional effects of AA restriction in health and disease.


Molecular Therapy | 2015

36. Genome-Wide Insight Into the Transcriptional Modulations Triggered By Lentiviral Transduction in Human Hematopoietic Stem Cells

Francesco Piras; Michela Riba; Dejan Lazarevic; Sara Bartolaccini; Elia Stupka; Davide Cittaro; Luigi Naldini; Anna Kajaste-Rudnitski

Recent studies suggest that hematopoietic stem cells (HSC) can sense foreign nucleic acids and pathogen-associated molecular patterns (PAMPs). Exposure to lentiviral vectors (LV) upon gene transfer may thus trigger acute host responses in HSC that could potentially impact on their biological properties, although no comprehensive studies are available to date. We have performed a high throughput RNA-Seq analysis on human cord-blood (CB)-derived CD34+ hematopoietic stem and progenitor cells (HSPC) exposed to research- or clinical-grade VSV-g pseudotyped (SIN) LV at a high multiplicity of infection, matching current clinical vector dose requirements. As controls, cells were exposed to non-transducing Env-less, genome-less or heat inactivated control vectors or kept in culture untreated. RNA was extracted at different times early after transduction, processed and ran in Illumina HiSeq2000. Analysis of Differential Expression in Time Course was performed using LIMMA R/BioConductor library. Key pathways were assessed by Term Enrichment Analysis considering KEGG pathways and Gene Ontology Biological processes. Transduction with both research-and clinical-grade LV significantly triggered DNA damage and apoptosis-related responses. In particular, p53 signaling was among the most significantly altered pathways (p<3.47×10−14) and induction of several key players, including a 8-fold increase in p21 mRNA, was further confirmed by Taqman. This signaling occurred also in bone-marrow-derived CD34+ cells and was integration-independent as Integrase-Defective LV (IDLV) induced p21 to a similar extent as LV. Furthermore, equal induction was observed in all CD34+ subpopulations, including in the most primitive CD38-CD133+ fraction. Finally, LV/IDLV exposure lead to a slight but significant increase in the percentage of apoptotic HSPC in culture (p<0.001) as compared to control vector exposed cells and untreated controls. Experiments are ongoing to further investigate the potential short and long-term consequences of this signaling on the biological properties of HSPC in vitro and in vivo. Overall, our results suggest for the first time that LV transduction triggers transcriptional changes in HSPC involving pathways pivotal for their biology. Better understanding of the potential functional consequences this may have will be important for the development of improved gene therapy protocols.

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Davide Cittaro

Vita-Salute San Raffaele University

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Elia Stupka

Vita-Salute San Raffaele University

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Dejan Lazarevic

Vita-Salute San Raffaele University

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Jose Manuel Garcia Manteiga

Vita-Salute San Raffaele University

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Luigi Naldini

Vita-Salute San Raffaele University

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Angelo Corti

Vita-Salute San Raffaele University

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Anna Kajaste-Rudnitski

Vita-Salute San Raffaele University

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Chiara Bonini

Vita-Salute San Raffaele University

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