A Two-Color Haploid Genetic Screen Identifies Novel Host Factors Involved in HIV-1 Latency

To identify novel host factors as putative targets to reverse HIV-1 latency, we performed an insertional mutagenesis genetic screen in a latent HIV-1 infected pseudohaploid KBM7 cell line (Hap-Lat). Following mutagenesis, insertions were mapped to the genome, and bioinformatic analysis resulted in the identification of 69 candidate host genes involved in maintaining HIV-1 latency. A select set of candidate genes was functionally validated using short hairpin RNA (shRNA)-mediated depletion in latent HIV-1 infected J-Lat A2 and 11.1 T cell lines. We confirmed ADK, CHD9, CMSS1, EVI2B, EXOSC8, FAM19A, GRIK5, IRF2BP2, NF1, and USP15 as novel host factors involved in the maintenance of HIV-1 latency. Chromatin immunoprecipitation assays indicated that CHD9, a chromodomain helicase DNA-binding protein, maintains HIV-1 latency via direct association with the HIV-1 5′ long terminal repeat (LTR), and its depletion results in increased histone acetylation at the HIV-1 promoter, concomitant with HIV-1 latency reversal. FDA-approved inhibitors 5-iodotubercidin, trametinib, and topiramate, targeting ADK, NF1, and GRIK5, respectively, were characterized for their latency reversal potential. While 5-iodotubercidin exhibited significant cytotoxicity in both J-Lat and primary CD4(+) T cells, trametinib reversed latency in J-Lat cells but not in latent HIV-1 infected primary CD4(+) T cells. Importantly, topiramate reversed latency in cell line models, in latently infected primary CD4(+) T cells, and crucially in CD4(+) T cells from three people living with HIV-1 (PLWH) under suppressive antiretroviral therapy, without inducing T cell activation or significant toxicity. Thus, using an adaptation of a haploid forward genetic screen, we identified novel and druggable host factors contributing to HIV-1 latency

Cytoskeletal Rearrangements in Synovial Fibroblasts as a Novel Pathophysiological Determinant of Modeled Rheumatoid Arthritis

Rheumatoid arthritis is a chronic inflammatory disease with a high prevalence and substantial socioeconomic burden. Despite intense research efforts, its aetiology and pathogenesis remain poorly understood. To identify novel genes and/or cellular pathways involved in the pathogenesis of the disease, we utilized a well-recognized tumour necrosis factor-driven animal model of this disease and performed high-throughput expression profiling with subtractive cDNA libraries and oligonucleotide microarray hybridizations, coupled with independent statistical analysis. This twin approach was validated by a number of different methods in other animal models of arthritis as well as in human patient samples, thus creating a unique list of disease modifiers of potential therapeutic value. Importantly, and through the integration of genetic linkage analysis and Gene Ontology–assisted functional discovery, we identified the gelsolin-driven synovial fibroblast cytoskeletal rearrangements as a novel pathophysiological determinant of the disease

Mutant KRAS promotes malignant pleural effusion formation

Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition

Soluble TNF Mediates the Transition from Pulmonary Inflammation to Fibrosis

BACKGROUND: Fibrosis, the replacement of functional tissue with excessive fibrous tissue, can occur in all the main tissues and organ systems, resulting in various pathological disorders. Idiopathic Pulmonary Fibrosis is a prototype fibrotic disease involving abnormal wound healing in response to multiple sites of ongoing alveolar epithelial injury. METHODOLOGY/PRINCIPAL FINDINGS: To decipher the role of TNF and TNF-mediated inflammation in the development of fibrosis, we have utilized the bleomycin-induced animal model of Pulmonary Fibrosis and a series of genetically modified mice lacking components of TNF signaling. Transmembrane TNF expression is shown to be sufficient to elicit an inflammatory response, but inadequate for the transition to the fibrotic phase of the disease. Soluble TNF expression is shown to be crucial for lymphocyte recruitment, a prerequisite for TGF-b1 expression and the development of fibrotic lesions. Moreover, through a series of bone marrow transfers, the necessary TNF expression is shown to originate from the non-hematopoietic compartment further localized in apoptosing epithelial cells. CONCLUSIONS: These results suggest a primary detrimental role of soluble TNF in the pathologic cascade, separating it from the beneficial role of transmembrane TNF, and indicate the importance of assessing the efficacy of soluble TNF antagonists in the treatment of Idiopathic Pulmonary Fibrosis

Molecular and genetic analysis of immunopathological mechanisms of chronic inflammation disorders

Inflammatory diseases are polygenic, widespread and life duration diseases, that lead to increased sickliness and influence more than the 20% of population of Europe. The last decade important progress in the comprehension of mechanisms of pathogenicity has been achieved but the precise concatenation and their interaction remains poorly understood. Via the rapid growth of functional genomics technologies, the correlation of phenotypic differences with by any chance changes in the expression of genome, allows the entanglement of concrete genes and/or cellular pathways in the event of particular phenotype. Objective is the localization of genes and paths of transfer of messages with sovereign role in the induction, the development and the severity of inflammatory diseases. With the systematic analysis of differential gene expression of two characteristic inflammatory diseases: the Rheumatoid Arthritis (RA) and the Idiopathic pulmonary fibrosis (IPF), we found differential expressed genes that contribute to disease pathogenesis. Those two phenotypically different diseases share a common cellular objective, the fibroblast. Rheumatoid Arthritis (RA) is a chronic devastating disease, which characterized by extended inflammation of joints that leads to the total destruction of bones and joints. Decisive role in the growth and event of the disease plays the fibroblast of synovial membrane. In the RA and under the pressure of inflammatory environment, the fibroblasts of synovial membrane are activated, with result the dramatic change of their gene expression. For the study of RA two differently animal experimental models were used, the hTNF and mTNFΔARE knock in. The differential gene expression of RA was analyzed with hybridization of oligonucleotide microarrays and subtractive DNA libraries in a twin highthrouput analysis. The data from each method processed with suitable analysis tools and the final lists were compared in order to create a common list of highly significant differentially expressed genes. The gene ontology (GO) analysis in the list of these statistically significant genes indicated the actin cytoskeleton as an important factor of pathogenesis of the disease, recomposition of which in the synovial fibroblasts is proposed as a new decisive pathophysiological factor of experimental RA. Also in the present work the significant role of the GSN gene in RA was revealed. Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive and usually mortal lung disease, of unknown etiology. Clinically, IPF is characterized by progressive, exertional dyspnea and nonproductive cough, worsening of pulmonary function and radiographically evident interstitial infiltrates (honeycombing). Histologically, IPF is associated with the appearance of Usual Interstitial Pneumonitis (UIP), which is characterized by patchy subpleural and/or paraseptal interstitial fibrosis alternating with areas of mild inflammation and normal lung. The hallmark of IPF/UIP is the distinctive presence of fibroblastic foci and exuberant Extracellular Matrix (ECM) deposition, leading to thickening of alveolar septa and the collapse of normal lung architecture. Patients that develop the fibrosis are usually individuals of medium age and independent sex. A 3% of cases it is presented in families with a background in the disease, thus declaring a genetic inclination...Οι φλεγμονώδεις παθήσεις είναι πολυγονιδιακές, ευρύτατα διαδεδομένες και δια βίου διάρκειας παθήσεις, που οδηγούν σε αυξημένη νοσηρότητα και επηρεάζουν περισσότερο από το 20% του πληθυσμού της Ευρώπης. Την τελευταία δεκαετία έχει επιτευχθεί σημαντική πρόοδος στην κατανόηση των μηχανισμών που διέπουν την παθογένεια τους, αλλά η ακριβής αλληλουχία και αλληλεπίδραση τους παραμένει εν πολλοίς άγνωστη. Μέσω της ραγδαίας ανάπτυξης των τεχνολογιών της λειτουργικής γονιδιωματικής, η συσχέτιση φαινοτυπικών διαφορών με τυχόν αλλαγές στην έκφραση του γονιδιώματος, επιτρέπει την εμπλοκή συγκεκριμένων γονιδίων ή/και κυτταρικών μονοπατιών στην εκδήλωση του συγκεκριμένου φαινοτύπου. Στόχος είναι ο εντοπισμός γονιδίων και μονοπατιών μεταβίβασης μηνυμάτων με κυρίαρχο ρόλο στην επαγωγή, στην εξέλιξη και στη βαρύτητα των φλεγμονωδών ασθενειών. Με τη συστηματική ανάλυση της διαφορικής γονιδιακής έκφρασης δύο χαρακτηριστικών φλεγμονωδών ασθενειών: της Ρευματοειδής Αρθρίτιδας (RA, Rheumatoid Arthritis) και της Ιδιοπαθούς Πνευμονικής Ίνωσης (IPF, Idiopathic Pulmonary Fibrosis, Usual interstitial pneumonia) βρέθηκαν γονίδια διαφορικά εκφρασμένα που ίσως εμπλέκονται στην παθογένεια της νόσου. Η δύο αυτές φαινοτυπικά διαφορετικές ασθένειες μοιράζονται ένα κοινό κυτταρικό στόχο, τον ινοβλάστη. Η Ρευματοειδής Αρθρίτιδα (ΡΑ) είναι μια χρόνια καταστροφική ασθένεια, που χαρακτηρίζεται από παρατεταμένη φλεγμονή των αρθρώσεων που οδηγεί στην ολική καταστροφή οστών και αρθρώσεων. Καθοριστικό ρόλο στην ανάπτυξη και εκδήλωση της νόσου διαδραματίζει ο ινοβλάστης αρθρικού υμένα. Στην ΡΑ και κάτω από την πίεση του φλεγμονώδους περιβάλλοντος, οι ινοβλάστες του αρθρικού υμένα ενεργοποιούνται, με αποτέλεσμα την δραματική αλλαγή της γονιδιακής τους έκφρασης. Για την μελέτη της Ρευματοειδής Αρθρίτιδας χρησιμοποιήθηκαν δύο διαφορετικά ζωικά πειραματικά πρότυπα, ο hTNF διαγονιδιακός και mTNFΔARE knock in ποντικός. Η διαφορική γονιδιακή έκφραση της ΡΑ αναλύθηκε με υβριδοποίηση ολιγονουκλεοτιδικών μικροσυστοιχιών και αφαιρετικών cDNA βιβλιοθηκών σε μία δίδυμη μεγάλου μεγέθους ανάλυση. Τα δεδομένα από κάθε μέθοδο επεξεργάστηκαν με κατάλληλα υπολογιστικά εργαλεία και οι τελικές λίστες συγκρίθηκαν για να δημιουργήσουν μια κοινή λίστα υψηλά σημαντικών διαφορικά εκφρασμένων γονιδίων. Η ανάλυση γονιδιακής οντολογίας στην λίστα των στατιστικά σημαντικών αυτών γονιδίων υπέδειξε εκτός των άλλων τον κυτταροσκελετό της ακτίνης ως ένα σημαντικό παράγοντα παθογένειας της νόσου, αναδιάταξη του οποίου στους αρθρικούς ινοβλάστες προτείνεται ως ένας νέος καθοριστικός παθοφυσιολογικός παράγοντας της πειραματικής ρευματοειδούς αρθρίτιδας. Επίσης στην παρούσα εργασία φανερώθηκε και ένας πρώιμος ρόλος του γονιδίου της GSN στην παθογένεια της ασθένειας. Η Ιδιοπαθής Πνευμονική Ίνωση (ΙΠΙ) είναι μια χρόνια, σταδιακή και συνήθως θανάσιμη πνευμονική πάθηση, αγνώστου αιτιολογίας.. Κλινικά χαρακτηρίζεται από δύσπνοια και μη παραγωγικό βήχα, ελαττωμένες πνευμονικές λειτουργίες και ραδιογραφικά προφανή διάχυτη διήθηση. Ιστολογικά χαρακτηρίζεται από διαφόρου βαθμού φλεγμονής ή/και ίνωσης, υπερπολλαπλασιασμό των διάμεσων ινοβλαστών/μυοινοβλαστών - οργανωμένων σε χαρακτηριστικά foci, υπερέκκριση συστατικών της εξωκυττάριας μήτρας (Extracellular matrix, ECM), απόπτωση των επιθηλιακών κυττάρων και απώλεια της φυσιολογικής αρχιτεκτονικής του πνεύμονα με κατάρρευση των αεροφόρων χώρων. Ασθενείς που αναπτύσσουν την ίνωση είναι συνήθως άτομα μέσης ηλικίας ανεξαρτήτου φύλου..

A Positive Regulatory Loop between a Wnt-Regulated Non-coding RNA and ASCL2 Controls Intestinal Stem Cell Fate

The canonical Wnt pathway plays a central role in stem cell maintenance, differentiation, and proliferation in the intestinal epithelium. Constitutive, aberrant activity of the TCF4/β-catenin transcriptional complex is the primary transforming factor in colorectal cancer. We identify a nuclear long non-coding RNA, termed WiNTRLINC1, as a direct target of TCF4/β-catenin in colorectal cancer cells. WiNTRLINC1 positively regulates the expression of its genomic neighbor ASCL2, a transcription factor that controls intestinal stem cell fate. WiNTRLINC1 interacts with TCF4/β-catenin to mediate the juxtaposition of its promoter with the regulatory regions of ASCL2. ASCL2, in turn, regulates WiNTRLINC1 transcriptionally, closing a feedforward regulatory loop that controls stem cell-related gene expression. This regulatory circuitry is highly amplified in colorectal cancer and correlates with increased metastatic potential and decreased patient survival. Our results uncover the interplay between non-coding RNA-mediated regulation and Wnt signaling and point to the diagnostic and therapeutic potential of WiNTRLINC1

Obesity Reshapes the Microbial Population Structure along the Gut-Liver-Lung Axis in Mice

The microbiome is emerging as a major player in tissue homeostasis in health and disease. Gut microbiome dysbiosis correlates with several autoimmune and metabolic diseases, while high-fat diets and ensuing obesity are known to affect the complexity and diversity of the microbiome, thus modulating pathophysiology. Moreover, the existence of a gut-liver microbial axis has been proposed, which may extend to the lung. In this context, we systematically compared the microbiomes of the gut, liver, and lung of mice fed a high-fat diet to those of littermates fed a matched control diet. We carried out deep sequencing of seven hypervariable regions of the 16S rRNA microbial gene to examine microbial diversity in the tissues of interest. Comparison of the local microbiomes indicated that lung tissue has the least diverse microbiome under healthy conditions, while microbial diversity in the healthy liver clustered closer to the gut. Obesity increased microbial complexity in all three tissues, with lung microbial diversity being the most modified. Obesity promoted the expansion of Firmicutes along the gut-liver-lung axis, highlighting staphylococcus as a possible pathologic link between obesity and systemic pathophysiology, especially in the lungs

The RNA-Binding Protein Elavl1/HuR Is Essential for Placental Branching Morphogenesis and Embryonic Development▿ †

HuR is an RNA-binding protein implicated in a diverse array of pathophysiological processes due to its effects on the posttranscriptional regulation of AU- and U-rich mRNAs. Here we reveal HuR's requirement in embryonic development through its genetic ablation. Obligatory HuR-null embryos exhibited a stage retardation phenotype and failed to survive beyond midgestation. By means of conditional transgenesis, we restricted HuR's mutation in either embryonic or endothelial compartments to demonstrate that embryonic lethality is consequent to defects in extraembryonic placenta. HuR's absence impaired the invagination of allantoic capillaries into the chorionic trophoblast layer and the differentiation of syncytiotrophoblast cells that control the morphogenesis and vascularization of the placental labyrinth and fetal support. HuR-null embryos rescued from these placental defects proceeded to subsequent developmental stages but displayed defects in skeletal ossification, fusions in limb elements, and asplenia. By coupling gene expression measurements, data meta-analysis, and HuR-RNA association assays, we identified transcription and growth factor mRNAs controlled by HuR, primarily at the posttranscriptional level, to guide morphogenesis, specification, and patterning. Collectively, our data demonstrate the dominant role of HuR in organizing gene expression programs guiding placental labyrinth morphogenesis, skeletal specification patterns, and splenic ontogeny