Mir-155-5p targets TP53INP1 to promote proliferative phenotype in hypersensitivity pneumonitis lung fibroblasts.

Background: Hypersensitivity pneumonitis (HP) is an inflammatory disorder affecting lung parenchyma and often evolves into fibrosis (fHP). The altered regulation of genes involved in the pathogenesis of the disease is not well comprehended, while the role of microRNAs in lung fibroblasts remains unexplored. Methods: We used integrated bulk RNA-Seq and enrichment pathway bioinformatic analyses to identify differentially expressed (DE)-miRNAs and genes (DEGs) associated with HP lungs. In vitro, we evaluated the expression and potential role of miR-155-5p in the phenotype of fHP lung fibroblasts. Loss and gain assays were used to demonstrate the impact of miR-155-5p on fibroblast functions. In addition, mir-155-5p and its target TP53INP1 were analyzed after treatment with TGF-ß, IL-4, and IL-17A. Results: We found around 50 DEGs shared by several databases that differentiate HP from control and IPF lungs, constituting a unique HP lung transcriptional signature. Additionally, we reveal 18 DE-miRNAs that may regulate these DEGs. Among the candidates likely associated with HP pathogenesis was miR-155-5p. Our findings indicate that increased miR-155-5p in fHP fibroblasts coincides with reduced TP53INP1 expression, high proliferative capacity, and a lack of senescence markers compared to IPF fibroblasts. Induced overexpression of miR-155-5p in normal fibroblasts remarkably increases the proliferation rate and decreases TP53INP1 expression. Conversely, miR-155-5p inhibition reduces proliferation and increases senescence markers. TGF-ß, IL-4, and IL-17A stimulated miR-155-5p overexpression in HP lung fibroblasts. Conclusion: Our findings suggest a distinctive signature of 53 DEGs in HP, including CLDN18, EEF2, CXCL9, PLA2G2D, and ZNF683, as potential targets for future studies. Likewise, 18 miRNAs, including miR-155-5p, could be helpful to establish differences between these two pathologies. The overexpression of miR-155-5p and downregulation of TP53INP1 in fHP lung fibroblasts may be involved in his proliferative and profibrotic phenotype. These findings may help differentiate and characterize their pathogenic features and understand their role in the disease.

Effect of Hypoxia in the Transcriptomic Profile of Lung Fibroblasts from Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by exacerbated extracellular matrix deposition that disrupts oxygen exchange. Hypoxia and its transcription factors (HIF-1α and 2α) influence numerous circuits that could perpetuate fibrosis by increasing myofibroblasts differentiation and by promoting extracellular matrix accumulation. Therefore, this work aimed to elucidate the signature of hypoxia in the transcriptomic circuitry of IPF-derived fibroblasts. To determine this transcriptomic signature, a gene expression analysis with six lines of lung fibroblasts under normoxia or hypoxia was performed: three cell lines were derived from patients with IPF, and three were from healthy donors, a total of 36 replicates. We used the Clariom D platform, which allows us to evaluate a huge number of transcripts, to analyze the response to hypoxia in both controls and IPF. The control's response is greater by the number of genes and complexity. In the search for specific genes responsible for the IPF fibroblast phenotype, nineteen dysregulated genes were found in lung fibroblasts from IPF patients in hypoxia (nine upregulated and ten downregulated). In this sense, the signaling pathways revealed to be affected in the pulmonary fibroblasts of patients with IPF may represent an adaptation to chronic hypoxia.

Lack of ZNF365 Drives Senescence and Exacerbates Experimental Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant activation of the alveolar epithelium, the expansion of the fibroblast population, and the accumulation of extracellular matrix. Global gene expression of human lung fibroblasts stimulated with TGFß-1, a strong fibrotic mediator revealed the overexpression of ZNF365, a zinc finger protein implicated in cell cycle control and telomere stabilization. We evaluated the expression and localization of ZNF365 in IPF lungs and in the fibrotic response induced by bleomycin in WT and deficient mice of the orthologous gene Zfp365. In IPF, ZNF365 was overexpressed and localized in fibroblasts/myofibroblasts and alveolar epithelium. Bleomycin-induced lung fibrosis showed an upregulation of Zfp365 localized in lung epithelium and stromal cell populations. Zfp365 KO mice developed a significantly higher fibrotic response compared with WT mice by morphology and hydroxyproline content. Silencing ZNF365 in human lung fibroblasts and alveolar epithelial cells induced a significant reduction of growth rate and increased senescence markers, including Senescence Associated ß Galactosidase activity, p53, p21, and the histone variant γH2AX. Our findings demonstrate that ZNF365 is upregulated in IPF and experimental lung fibrosis and suggest a protective role since its absence increases experimental lung fibrosis mechanistically associated with the induction of cell senescence.

Cyclic Attractors Are Critical for Macrophage Differentiation, Heterogeneity, and Plasticity.

Adaptability, heterogeneity, and plasticity are the hallmarks of macrophages. How these complex properties emerge from the molecular interactions is an open question. Thus, in this study we propose an actualized regulatory network of cytokines, signaling pathways, and transcription factors to survey the differentiation, heterogeneity, and plasticity of macrophages. The network recovers attractors, which in regulatory networks correspond to cell types, that correspond to M0, M1, M2a, M2b, M2c, M2d, M2-like, and IL-6 producing cells, including multiple cyclic attractors that are stable to perturbations. These cyclic attractors reproduce experimental observations and show that oscillations result from the structure of the network. We also study the effect of the environment in the differentiation and plasticity of macrophages, showing that the observed heterogeneity in macrophage populations is a result of the regulatory network and its interaction with the micro-environment. The macrophage regulatory network gives a mechanistic explanation to the heterogeneity and plasticity of macrophages seen in vivo and in vitro, and offers insights into the mechanism that allows the immune system to react to a complex dynamic environment.

CD4+ T Cell Regulatory Network Underlies the Decrease in Th1 and the Increase in Anergic and Th17 Subsets in Severe COVID-19.

In this model we use a dynamic and multistable Boolean regulatory network to provide a mechanistic explanation of the lymphopenia and dysregulation of CD4+ T cell subsets in COVID-19 and provide therapeutic targets. Using a previous model, the cytokine micro-environments found in mild, moderate, and severe COVID-19 with and without TGF-ß and IL-10 was we simulated. It shows that as the severity of the disease increases, the number of antiviral Th1 cells decreases, while the the number of Th1-like regulatory and exhausted cells and the proportion between Th1 and Th1R cells increases. The addition of the regulatory cytokines TFG-ß and IL-10 makes the Th1 attractor unstable and favors the Th17 and regulatory subsets. This is associated with the contradictory signals in the micro-environment that activate SOCS proteins that block the signaling pathways. Furthermore, it determined four possible therapeutic targets that increase the Th1 compartment in severe COVID-19: the activation of the IFN-γ pathway, or the inhibition of TGF-ß or IL-10 pathways or SOCS1 protein; from these, inhibiting SOCS1 has the lowest number of predicted collateral effects. Finally, a tool is provided that allows simulations of specific cytokine environments and predictions of CD4 T cell subsets and possible interventions, as well as associated secondary effects.

Inflammatory pathways are upregulated in the nasal epithelium in patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive scarring of the lung parenchyma, leading to respiratory failure and death. High resolution computed tomography of the chest is often diagnostic for IPF, but its cost and the risk of radiation exposure limit its use as a screening tool even in patients at high risk for the disease. In patients with lung cancer, investigators have detected transcriptional signatures of disease in airway and nasal epithelial cells distal to the site of disease that are clinically useful as screening tools. Here we assessed the feasibility of distinguishing patients with IPF from age-matched controls through transcriptomic profiling of nasal epithelial curettage samples, which can be safely and repeatedly sampled over the course of a patient's illness. We recruited 10 patients with IPF and 23 age-matched healthy control subjects. Using 3' messenger RNA sequencing (mRNA-seq), we identified 224 differentially expressed genes, most of which were upregulated in patients with IPF compared with controls. Pathway enrichment analysis revealed upregulation of pathways related to immune response and inflammatory signaling in IPF patients compared with controls. These findings support the concept that fibrosis is associated with upregulation of inflammatory pathways across the respiratory epithelium with possible implications for disease detection and pathobiology.

Improving biocuration of microRNAs in diseases: a case study in idiopathic pulmonary fibrosis.

MicroRNAs (miRNAs) are small and non-coding RNA molecules that inhibit gene expression posttranscriptionally. They play important roles in several biological processes, and in recent years there has been an interest in studying how they are related to the pathogenesis of diseases. Although there are already some databases that contain information for miRNAs and their relation with illnesses, their curation represents a significant challenge due to the amount of information that is being generated every day. In particular, respiratory diseases are poorly documented in databases, despite the fact that they are of increasing concern regarding morbidity, mortality and economic impacts. In this work, we present the results that we obtained in the BioCreative Interactive Track (IAT), using a semiautomatic approach for improving biocuration of miRNAs related to diseases. Our procedures will be useful to complement databases that contain this type of information. We adapted the OntoGene text mining pipeline and the ODIN curation system in a full-text corpus of scientific publications concerning one specific respiratory disease: idiopathic pulmonary fibrosis, the most common and aggressive of the idiopathic interstitial cases of pneumonia. We curated 823 miRNA text snippets and found a total of 246 miRNAs related to this disease based on our semiautomatic approach with the system OntoGene/ODIN. The biocuration throughput improved by a factor of 12 compared with traditional manual biocuration. A significant advantage of our semiautomatic pipeline is that it can be applied to obtain the miRNAs of all the respiratory diseases and offers the possibility to be used for other illnesses. Database URL: http://odin.ccg.unam.mx/ODIN/bc2015-miRNA/.

Assisted curation of regulatory interactions and growth conditions of OxyR in E. coli K-12.

Given the current explosion of data within original publications generated in the field of genomics, a recognized bottleneck is the transfer of such knowledge into comprehensive databases. We have for years organized knowledge on transcriptional regulation reported in the original literature of Escherichia coli K-12 into RegulonDB (http://regulondb.ccg.unam.mx), our database that is currently supported by >5000 papers. Here, we report a first step towards the automatic biocuration of growth conditions in this corpus. Using the OntoGene text-mining system (http://www.ontogene.org), we extracted and manually validated regulatory interactions and growth conditions in a new approach based on filters that enable the curator to select informative sentences from preprocessed full papers. Based on a set of 48 papers dealing with oxidative stress by OxyR, we were able to retrieve 100% of the OxyR regulatory interactions present in RegulonDB, including the transcription factors and their effect on target genes. Our strategy was designed to extract, as we did, their growth conditions. This result provides a proof of concept for a more direct and efficient curation process, and enables us to define the strategy of the subsequent steps to be implemented for a semi-automatic curation of original literature dealing with regulation of gene expression in bacteria. This project will enhance the efficiency and quality of the curation of knowledge present in the literature of gene regulation, and contribute to a significant increase in the encoding of the regulatory network of E. coli. RegulonDB Database URL: http://regulondb.ccg.unam.mx OntoGene URL: http://www.ontogene.org.

Transcription factors in Escherichia coli prefer the holo conformation.

The transcriptional regulatory network of Escherichia coli K-12 is among the best studied gene networks of any living cell. Transcription factors bind to DNA either with their effector bound (holo conformation), or as a free protein (apo conformation) regulating transcription initiation. By using RegulonDB, the functional conformations (holo or apo) of transcription factors, and their mode of regulation (activator, repressor, or dual) were exhaustively analyzed. We report a striking discovery in the architecture of the regulatory network, finding a strong under-representation of the apo conformation (without allosteric metabolite) of transcription factors when binding to their DNA sites to activate transcription. This observation is supported at the level of individual regulatory interactions on promoters, even if we exclude the promoters regulated by global transcription factors, where three-quarters of the known promoters are regulated by a transcription factor in holo conformation. This genome-scale analysis enables us to ask what are the implications of these observations for the physiology and for our understanding of the ecology of E. coli. We discuss these ideas within the framework of the demand theory of gene regulation.