Identification of a miR-146b-FasL axis in the development of neutropenia in T large granular lymphocyte leukemia

T Large Granular Lymphocytes leukemia is characterized by the expansion of several Large Granular Lymphocyte clones, among which a subset of Large Granular Lymphocytes showing constitutively activated STAT3, a specific CD8+/CD4- phenotype and the presence of neutropenia has been identified. Although STAT3 is an inducer of transcription of a large number of oncogenes, so far its relationship with miRNA has not been evaluated in T-Large Granular Lymphocyte Leukemia patients. Here, we investigated whether STAT3 could carry out its pathogenetic role in T-Large Granular Lymphocyte Leukemia through an altered expression of miRNAs. The expression level of 756 mature miRNAs was assessed on purified T-LGLs by using a TaqMan Human microRNA Array. Hierarchical Clustering Analysis of miRNA array data shows that the global miRNome clusters with CD8 T-Large Granular Lymphocytes. Remarkably, CD8 T-Large Granular Lymphocytes exhibit a selective and STAT3-dependent repression of miR-146b expression, that significantly correlated with the absolute neutrophil counts and inversely correlated with the expression of FasL, that is regarded as the most relevant factor in the pathogenesis of neutropenia. Experimental evidence demonstrates that the STAT3-dependent reduction of miR-146b expression in CD8 T-Large Granular Lymphocytes occurs as a consequence of miR-146b promoter hypermethylation and results in the disruption of the HuR-mediated post-transcriptional machinery controlling FasL mRNA stabilization. Restoring miR-146b expression in CD8 T-Large Granular Lymphocytes lead to a reduction of HuR protein and, in turn, of FasL mRNA expression, thus providing mechanistic insights for the existence of a STAT3-miR146b-FasL axis and neutropenia in T-Large Granular Lymphocyte Leukemia

Analyzing BioRad-Illumina Single Cell RNA-Seq data with open source tools

Single cell RNA-Seq is a powerful technique that is becoming more popular since it enables to sequence the transcriptome of each cell within a population of different cell types in a single experiment. Currently, there are a few different technologies, like BioRad-Illumina ddSeq and 10X Chromium

Chromatin remodelling and autocrine TNFα are required for optimal interleukin-6 expression in activated human neutrophils

How IL-6 expression is regulated in human neutrophils has remained unclear. Here the authors show, using highly purified neutrophils, that TLR8 or TLR4 signalling activates latent enhancers and cooperates with autocrine TNFα to induce IL-6 transcription

Anti-viral state segregates two molecular phenotypes of pancreatic adenocarcinoma: potential relevance for adenoviral gene therapy

<p>Abstract</p> <p>Background</p> <p>Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer mortality for which novel gene therapy approaches relying on tumor-tropic adenoviruses are being tested.</p> <p>Methods</p> <p>We obtained the global transcriptional profiling of primary PDAC using RNA from eight xenografted primary PDAC, three primary PDAC bulk tissues, three chronic pancreatitis and three normal pancreatic tissues. The Affymetrix GeneChip HG-U133A was used. The results of the expression profiles were validated applying immunohistochemical and western blot analysis on a set of 34 primary PDAC and 10 established PDAC cell lines. Permissivity to viral vectors used for gene therapy, Adenovirus 5 and Adeno-Associated Viruses 5 and 6, was assessed on PDAC cell lines.</p> <p>Results</p> <p>The analysis of the expression profiles allowed the identification of two clearly distinguishable phenotypes according to the expression of interferon-stimulated genes. The two phenotypes could be readily recognized by immunohistochemical detection of the Myxovirus-resistance A protein, whose expression reflects the activation of interferon dependent pathways. The two molecular phenotypes discovered in primary carcinomas were also observed among established pancreatic adenocarcinoma cell lines, suggesting that these phenotypes are an intrinsic characteristic of cancer cells independent of their interaction with the host's microenvironment. The two pancreatic cancer phenotypes are characterized by different permissivity to viral vectors used for gene therapy, as cell lines expressing interferon stimulated genes resisted to Adenovirus 5 mediated lysis in vitro. Similar results were observed when cells were transduced with Adeno-Associated Viruses 5 and 6.</p> <p>Conclusion</p> <p>Our study identified two molecular phenotypes of pancreatic cancer, characterized by a differential expression of interferon-stimulated genes and easily recognized by the expression of the Myxovirus-resistance A protein. We suggest that the detection of these two phenotypes might help the selection of patients enrolled in virally-mediated gene therapy trials.</p

Histological verification of positive positron emission tomography findings in the follow-up of patients with mediastinal lymphoma.

Background and Objectives Follow-ups of patients with mediastinal lymphoma are not accurate if they rely on computed tomography (CT). Positron emission tomography (PET) has been suggested to be useful in several lymphoma settings, such as initial staging, evaluation of residual masses after therapy, and assessment of response early in the course of treatment. The aim of this retrospective study was to verify the reliability of positive PET scans of the mediastinum in following up patients wirh mediastinal lymphoma, using histological findings as a comparison. Design and Methods From January 2002 to July 2005, 151 patients with mediastinal lymphoma (57 with Hodgkin's disease [HD] and 94 with aggressive non-Hodgkin's lymphoma [NHL]) were followed-up after the end of front-line treatment. Patients with a positive PET scan of the mediastinum underwent CT scanning and surgical biopsy. Results In 30 (21 HD and 9 NHL) out of 151 patients (20%) a suspicion of lymphoma relapse was raised based on positive mediastinal PET scanning. Histology confirmed this suspicion in 17 (10 HD and 7 NHL) out of 30 patients (57%), whereas either benign (9 fibrosis, 3 sarcoid-like granulomatosis) or unrelated neoplastic conditions (1 thymoma) were demonstrated in the remaining 13 patients (43%). SUVmax was significantly higher among patients who had signs of relapse (17 true positive cases) than among those who stayed in remission (13 false positive cases), the median values being 5.95 (range, 3.5–26.9) and 2.90 (range, 1.4–3.3), respectively ( p =0.01). Interpretation and Conclusions We suggest that a positive PET scan of the mediastinum of a patient being followed-up for a mediastinal lymphoma should not be considered sufficient for diagnostic purposes in view of its lack of discrimination. Histological confirmation can safely be carried out with various biopsy techniques, the choice of which should be made on the basis of the findings of the clinical and imaging studies of the individual case

Histone modifications underlie monocyte dysregulation in patients with systemic sclerosis, underlining the treatment potential of epigenetic targeting.

Background and objective S ystemic sclerosis (SSc) is a severe autoimmune disease, in which the pathogenesis is dependent on both genetic and epigenetic factors. Altered gene expression in SSc monocytes, particularly of interferon (IFN)-responsive genes, suggests their involvement in SSc development. We investigated the correlation between epigenetic histone marks and gene expression in SSc monocytes. Methods C hromatin immunoprecipitation followed by sequencing (ChIPseq) for histone marks H3K4me3 and H3K27ac was performed on monocytes of nine healthy controls and 14 patients with SSc. RNA sequencing was performed in parallel to identify aberrantly expressed genes and their correlation with the levels of H3K4me3 and H3K27ac located nearby their transcription start sites. ChIP-qPCR assays were used to verify the role of bromodomain proteins, H3K27ac and STATs on IFNresponsive gene expression. Results 1046 and 534 genomic loci showed aberrant H3K4me3 and H3K27ac marks, respectively, in SSc monocytes. The expression of 381 genes was directly and significantly proportional to the levels of such chromatin marks present near their transcription start site. Genes correlated to altered histone marks were enriched for immune, IFN and antiviral pathways and presented with recurrent binding sites for IRF and STAT transcription factors at their promoters. IFN\u3b1 induced the binding of STAT1 and STAT2 at the promoter of two of these genes, while blocking acetylation readers using the bromodomain BET family inhibitor JQ1 suppressed their expression. Conclusion SS c monocytes have altered chromatin marks correlating with their IFN signature. Enzymes modulating these reversible marks may provide interesting therapeutic targets to restore monocyte homeostasis to treat or even prevent SSc

Preclinical evaluation of KIT/PDGFRA and mTOR inhibitors in gastrointestinal stromal tumors using small animal FDG PET

<p>Abstract</p> <p>Background</p> <p>Primary and secondary drug resistance to imatinib and sunitinib in patients with gastrointestinal stromal tumors (GISTs) has led to a pressing need for new therapeutic strategies such as drug combinations. Most GISTs are caused by mutations in the KIT receptor, leading to upregulated KIT tyrosine kinase activity. Imatinib and nilotinib directly inhibit the kinase activity of KIT, while RAD001 (everolimus) inhibits mTOR. We report a preclinical study on drug combinations in a xenograft model of GIST in which effects on tumor dimensions and metabolic activity were assessed by small animal PET imaging.</p> <p>Methods</p> <p>Rag2-/-; γcommon -/- male mice were injected s.c. into the right leg with GIST 882. The animals were randomized into 6 groups of 6 animals each for different treatment regimens: No therapy (control), imatinib (150 mg/kg b.i.d.) by oral gavage for 6 days, then once/day for another 7 days, everolimus (10 mg/kg/d.) by oral gavage, everolimus (10 mg/kg/d.) + imatinib (150 mg/kg b.i.d.) by oral gavage for 6 days, then once/day for another 7 days, nilotinib (75 mg/kg/d.) by oral gavage, nilotinib (75 mg/kg/d.) + imatinib (150 mg/kg b.i.d) by oral gavage for 6 days, then once/day for another 7 days. Tumor growth control was evaluated by measuring tumor volume (cm³). Small animal PET (GE Explore tomography) was used to evaluate tumor metabolism and performed in one animal per group at base-line then after 4 and 13 days of treatment.</p> <p>Results</p> <p>After a median latency time of 31 days, tumors grew in all animals (volume 0,06-0,15 cm³) and the treatments began at day 38 after cell injection. Tumor volume control (cm3) after 13 days of treatment was > 0.5 for imatinib alone and nilotinib alone, and < 0.5 for the 2 combinations of drugs and for everolimus alone. The baseline FDG uptake was positive in all animals. FDG/SUV/TBR was strongly reduced over time by everolimus both as a single agent and in combination with imatinib respectively: 3.1 vs. 2.3 vs. 1.9 and 2.5 vs 2.3 vs 0.</p> <p>Conclusions</p> <p>As single agents, all drugs showed an anti-tumor effect in GIST xenografts but everolimus was superior. The everolimus plus imatinib combination appeared to be the most active regimen both in terms of inhibiting tumor growth and tumor metabolism. The integration of everolimus in GIST treatment merits further investigation.</p

Modulation of lipopolysaccharide-induced gene transcription by IL-10: focus on transcription factors and chromatin modifications.

Il processo infiammatorio \ue8 costituito dalla successione temporale di eventi coordinati dalla presenza di distinte citochine pro- e antinfiammatorie. Il mancato controllo temporale e/o dell'entit\ue0 della risposta infiammatoria pu\uf2 svolgere un ruolo importante nello sviluppo di patologie infiammatorie croniche e autoimmuni. In questo contesto, la corretta evoluzione dell\u2019infiammazione, finalizzata all\u2019eliminazione di agenti patogeni senza generare danno ai tessuti, \ue8 fortemente condizionata dai livelli relativi di citochine pro- e antiinfiammatorie. Per questo la caratterizzazione delle vie di trasduzione del segnale che regolano l\u2019espressione dei geni codificanti per citochine durante l\u2019infiammazione \ue8 di particolare importanza, ed ha rilevanti ricadute traslazionali. L\u2019entit\ue0 e la durata della risposta infiammatoria sono finemente regolate dall\u2019 Interleuchina (IL)-10, una potente citochina antinfiammatoria, in grado di limitare l\u2019espressione e la produzione di citochine e chemochine proinfiammatorie e di promuovere il rilascio di molecole antinfiammatorie da monociti, macrofagi e neutrofili attivati in seguito a stimolazione dei recettori Toll-like (TLRs), come il lipopolisaccaride (LPS) batterico. Nonostante l\u2019IL-10 sia una citochina molto studiata per le sue attivit\ue0 antinfiammatorie ed immunomodulatorie, il meccanismo molecolare attraverso cui esercita le sue azioni biologiche \ue8 ancora controverso. Lo scopo di questa tesi \ue8 quello di caratterizzare i meccanismi molecolari attraverso i quali l\u2019IL-10 modula l\u2019espressione di citochine infiammatorie in monociti e neutrofili umani attivati dall\u2019LPS. In particolare lo studio si \ue8 articolato in modo da perseguire due obiettivi principali: (A) Caratterizzazione dei meccanismi molecolari attraverso cui IL-10 potenzia la trascrizione del mediatore anti-infiammatoria antagonista del recettore per Interleuchina 1 (IL-1ra) indotta da LPS; (B) Caratterizzazione dei meccanismi molecolari attraverso cui IL-10 inibisce la trascrizione di due mediatori proinfiammatori indotti da LPS, la chemochina CXCL8 e la citochina TNF-a pro-infiammatorie indotte da LPS. (A) I nostri risultati dimostrano che nei monociti e neutrofili stimolati con LPS, NF-kBp65 e NF-kBp50 traslocano nel nucleo e vengono reclutati sul promotore di geni NF-kB-dipendenti (come ad es. IkBa), ma non sul promotore di IL-1ra, nonostante questo possieda dei siti funzionali di legame per NF-kB. Tale reclutamento diviene efficiente solo in presenza di IL-10. Infatti, IL-10, promuovendo l\u2019acetilazione della cromatina a livello del promotore di IL-1ra, smaschera i siti di legame favorendo cos\uec il reclutamento di NF-kBp65 e NF-kBp50. Questo processo \ue8 strettamente dipendente dall\u2019attivazione di STAT3 da parte dell\u2019IL-10, come dimostrato in neutrofili purificati da pazienti affetti da hyper IgE (HIES) che hanno uno STAT3 non funzionale. In queste cellule, la mancata attivazione di STAT3 da parte dell\u2019IL-10 impedisce sia l\u2019induzione del reclutamento di NF-kBp65 e NF-kBp50 sul promotore di IL-1ra che il potenziamento della trascrizione di questo gene. Il nostro studio descrive per la prima volta come IL-10, utilizzando un meccanismo che modifica la struttura della cromatina, moduli l\u2019espressione di IL-1ra indotta da LPS in monociti e neutrofili umani. La rilevanza di tale meccanismo \ue8 supportata dalle osservazioni fatte in pazienti HIES, in cui la difettosa attivazione di STAT3 indotta da IL-10 \ue8 responsabile di una risposta immunitaria innata eccessiva. (B) Nella seconda parte della tesi abbiamo analizzato i meccanismi molecolari attraverso cui l\u2019 IL-10 inibisce l\u2019espressione di citochine pro-infiammatorie indotta da LPS. Lo studio \ue8 focalizzato su due classici ed importanti mediatori proinfiammatori: la chemochina CXCL8 e la citochina TNF-a. I dati ottenuti indicano che IL-10 utilizza meccanismi diversi e cellulo-specifici per inibire la trascrizione di CXCL8 e TNF-a indotta da LPS. Infatti, IL-10 inibisce direttamente il reclutamento della Polimerasi II (Pol II), indotto da LPS, sui promotori di CXCL8 e TNF-a nei neutrofili, ma non nei monociti. La riduzione del reclutamento della Pol II nei neutrofili correla con la riduzione dei livelli di c-fos reclutati in seguito a stimolazione con LPS sui promotori di CXCL8 e TNF-a. Questa osservazione suggerisce che i due eventi siano causalmente correlati. Al contrario, nei monociti, IL-10 inibisce il reclutamento dalla ciclina chinasi 9 (CDK9) e di conseguenza l\u2019attivazione (cio\ue8 la fosforilazione in Serina-2) della Pol II legata ai promotori, inibendo in questo modo l\u2019allungamento del trascritto. L\u2019inibizione del reclutamento della CDK9 avviene con modalit\ue0 diverse per ciascun promotore. Infatti, nel caso del promotore di CXCL8, l\u2019IL-10, probabilmente attraverso il reclutamento e/o attivazione di una istone-deacetilasi (HDAC), riduce i livelli di acetilazione dell\u2019istone H4 associato al promotore di CXCL8. Questa riduzione determina un mancato reclutamento di Brd4 e, di conseguenza, di CDK9. Nel caso del TNF-a, invece, l\u2019inibizione del reclutamento di CDK9 \ue8 probabilmente dovuta all\u2019inibizione del reclutamento di c-Jun indotto dall\u2019LPS. Complessivamente, questi dati portano alla luce un nuovo meccanismo utilizzato da IL-10 per inibire la trascrizione di CXCL8 e TNF-a.The inflammatory process consists of a coordinated, sequential and self-limiting release of different mediators that orchestrate and control the chronological phases of leukocytes recruitment, activation, and clearance. In this contest, it is well known that the appropriate balance between pro- and anti-inflammatory cytokines production and action is essential for successful innate immune response that clears infectious pathogens but limits tissue damage and autoimmunity. The characterization of the pathways and checkpoints that regulate cytokine gene expression during physiologic inflammation is of particular importance, since it will yield insights into fundamental events occurring in disorders characterized by deregulated inflammation. The amplitude and the duration of the inflammatory response are fine-tuned by Interleukin (IL)-10, a potent antinflammatory cytokine produced, among others, by myeloid cells in response to Toll-like receptor (TLR) activation and, in turn, very effective at suppressing TLR-induced gene expression and inflammatory cytokine production. Although IL-10 antinflammatory properties have been thoroughly described, the molecular mechanisms governing IL-10 production and actions on cells of the innate immune system have not been fully elucidated yet. The purpose of the thesis is to provide a comprehensive characterization of the molecular mechanisms through which IL-10 modulates inflammatory cytokine gene expression in human monocytes and neutrophils exposed to lipopolysaccharide (LPS). In particular, the study is focused on two main goals: (A) characterization of the molecular mechanisms through which IL-10 potentiate the transcription of the LPS-induced anti-inflammatory mediator, the Interleukin 1 receptor antagonist (IL-1ra); (B) characterization of the molecular mechanisms through which IL-10 inhibits the LPS-induced transcription of two pro-inflammatory mediators, a chemokine, CXCL8 and a cytokine, TNF-a. (A) Our results show that in monocytes and neutrophils while NF-kBp65 and NF-kBp50 proteins accumulate into the nuclei and bind to the promoter of IkBa during LPS stimulation, they are not recruited to the kB sites of the IL-1ra promoter. However, in the presence of LPS plus IL-10, which we found to induce chromatin acetylation, recruitment of both NF-kBp65 and NF-kBp50 to the IL-1ra promoter efficiently occurs in a STAT3-dependent manner. Accordingly, a failure of IL-10 to promote NF-kBp65 recruitment to the IL-1ra promoter, and consequently to potentiate LPS-induced IL-1ra transcription, was observed in neutrophils from hyper IgE syndrome (HIES) patients, who carry a non-functional STAT3. Our study uncovers a gene-specific targeting of chromatin structure as a previously undescribed mechanisms used by IL-10 to modulate LPS-induced IL-1ra expression in human monocytes and neutrophils. The relevance of such mechanism is supported by the observations made in HIES patients, in whom the defective IL-10-induced activation of STAT3 is responsible for an overly activated innate immune responses. (B) In the second part of the thesis we addressed the question how IL-10 does inhibit LPS-induced pro-inflammatory gene transcription, focusing the study on CXCL8 and TNF-a. Using quantitative chromatin immunoprecipitation (ChIP) analysis, we characterized the molecular events triggered by IL-10 at the level of CXCL8 and/or TNF-a gene promoters. Our data show that IL-10 triggers cell type specific mechanisms to inhibit LPS-induced CXCL8 and TNF-a transcription. In fact, IL-10 inhibits LPS-induced recruitment of Pol II to the CXCL8 and TNF-a promoter in neutrophils, but not in monocytes. Reduction of Pol II recruitment to the CXCL8 and TNF-a promoters in neutrophils correlates with a reduction of the levels of LPS-induced c-fos recruitment to the same promoters, thus suggesting a cause-effect relationship between these two events. Conversely, in monocytes, IL-10 reduces the recruitment of CDK9 induced by LPS and, as a consequence, inhibits LPS-induced activation (i.e. ser-2-phosphorylation) of promoter-bound Pol II. Inhibition of CDK9 recruitment by IL-10 proceeds in a gene specific manner. In fact, in the case of CXCL8, IL-10, via the recruitment and/or activation of an histone deacethylase, reduces the levels of acetylated histone H4. This results into the inhibition of Brd4 and, subsequently, of the Pol II activating Cyclin kinase 9 recruitment to the CXCL8 promoter. In the case of TNF-a, the decrease in promoter-bound CDK9 is likely to be dependent on IL-10-mediated reduction of LPS-induced association of c-Jun to the TNF-a promoter. Collectively, our data unravel novel and cell type-specific mechanisms utilized by IL-10 to inhibit LPS-induced CXCL8 and TNF-a transcription

Identification and characterization of Long non-coding RNAs in primary human monocytes and neutrophils

Long non-coding RNAs (LncRNAs) are described as key players in several biological and pathological processes thanks to their ability to regulate gene expression [1]. The role of LncRNAs in development and cancer has been deeply investigated [2]. Conversely, few is known about LncRNAs in immune response [3]. With this background, our study aims to identify and characterize the LncRNAs in human neutrophils (PMN) and monocytes responses to TLR4 activation. RNA seq was performed on human CD14+ monocytes and highly-pure PMN stimulated with LPS for 90min and 4h or left untreated. Differential gene expression was analysed by DESeq2. LPS is able both to induce the de novo expression and to modulate constitutively expressed LncRNAs in monocytes and PMN. Further analysis of the 2278 LncRNAs regulated by LPS in both cell types allowed us to identify: i)626 LncRNAs commonly modulated by LPS in both phagocyte types; ii)1186 LncRNAs modulated uniquely in monocytes, and 466 modulated selectively in PMN. Moreover, K-mean clustering analysis identifies three groups of LncRNAs differently modulated by LPS: Early: LncRNAs modulated by LPS already at 90m; Early and transient: rapidly modulated LncRNAs whose expression returns to the basal level by 4h; Late: LncRNAs modulated by LPS after 4h. Moreover, eRNA and canonical LncRNAs were identified based on H3K4me3 and H3K4me1 ChIP-seq analysis. Finally, to get insight into LncRNAs function, protein coding genes in cis to LncRNAs were analysed for GO Term enrichment. This in silico analysis suggests a likely role for LncRNAs in the regulation of several phagocyte processes, among which the most representative are cytokine production, signal transduction, chromatin organization and metabolic processes. Our study shows that LncRNAs are modulated and are likely involved in the regulation of many biological processes in human PMN and monocytes triggered by TLR4 activation. The detail functional characterization of this class of regulators is under investigation

Characterization of NRIR function in LPS treated human CD14+ monocytes.

The aim of this study is to identify the role of NRIR in LPS treated human CD14+ monocytes. RNAseq analysis of LPS treated primary human CD14+ monocytes and autologous neutrophils (PMN) allowed us to classify lncRNAs modulated by LPS in a cell type specific manner. Specifically 1186 lncRNAs modulated by LPS in a monocyte specific manner, whereas 466 lncRNAs selectively modulated by LPS only in PMN. Among the monocyte-specific lncRNAs we focused our attention on NRIR for the following reasons: NRIR is strongly upregulated by LPS in monocytes; NRIR was described as an interferon (IFN) dependent lncRNA able to negative regulate the expression of IFN-dependent genes in hepatoma cell line; in PMN, LPS does not trigger the interferon response nor it upregulates NRIR expression, thus suggesting that the two events might be causally linked; the role of NRIR in inflammation and particularly in monocytes response to LPS remains to be elucidated. Kinetic analysis showed that NRIR expression starts to be detectable in CD14+ monocytes 4 hours post LPS (100ng/mL) stimulation, and keeps increasing up to 16 hours. Remarkably, IFNa (1000 U/mL) is a stronger and more rapid inducer of NRIR expression, that starts at 2h and keeps increasing up to 16h. Subcellular fractionation of LPS-treated monocytes showed that NRIR localizes in the nuclear fraction. To gain insights into NRIR function, RNAseq data of LPS treated CD14+ monocytes were used for the Weighted Gene Co-expression Network Analysis (WGCNA) and the NRIR specific WGCNA module was created and analyzed. GO term analysis of the protein coding genes of NRIR module displays an enrichment of genes involved in several processes, among which type I IFN and viral response was of particular interest. Taken together, these experimental and in silico data hint for a possible of NRIR in the type I IFN response triggered by LPS in human monocytes. This hypothesis is currently under investigation by loss of function strategy