CSF1R inhibition by small molecule affects T-helper cell differentiation independently of microglia depletion

Colony-stimulating factor 1 receptor (CSF1R) inhibition has been proposed as a specific method for microglia depletion. However, recent work revealed that in addition to microglia, CSF1R inhibition also affects other innate immune cells, such as peripheral monocytes and tissue-resident macrophages of the lung, liver, spleen, and peritoneum. Here, we show that this effect is not restricted to innate immune cells only, but extends to the adaptive immune compartment. CSF1R inhibition alters the transcriptional profile of bone marrow cells that control T helper cell activation. In vivo or ex vivo inhibition of CSF1R profoundly changes the transcriptional profile of CD4+ cells and suppresses Th1 and Th2 differentiation in directionally stimulated and unstimulated cells and independently of microglia depletion. Given that T cells also contribute in CNS pathology, these effects may have practical implications in the interpretation of relevant experimental data.

Transcriptional Profiling and Machine Learning Unveil a Concordant Biosignature of Type I Interferon-Inducible Host Response Across Nasal Swab and Pulmonary Tissue for COVID-19 Diagnosis

BackgroundCOVID-19, caused by SARS-CoV-2 virus, is a global pandemic with high mortality and morbidity. Limited diagnostic methods hampered the infection control. Since the direct detection of virus mainly by RT-PCR may cause false-negative outcome, host response-dependent testing may serve as a complementary approach for improving COVID-19 diagnosis.ObjectiveOur study discovered a highly-preserved transcriptional profile of Type I interferon (IFN-I)-dependent genes for COVID-19 complementary diagnosis.MethodsComputational language R-dependent machine learning was adopted for mining highly-conserved transcriptional profile (RNA-sequencing) across heterogeneous samples infected by SARS-CoV-2 and other respiratory infections. The transcriptomics/high-throughput sequencing data were retrieved from NCBI-GEO datasets (GSE32155, GSE147507, GSE150316, GSE162835, GSE163151, GSE171668, GSE182569). Mathematical approaches for homological analysis were as follows: adjusted rand index-related similarity analysis, geometric and multi-dimensional data interpretation, UpsetR, t-distributed Stochastic Neighbor Embedding (t-SNE), and Weighted Gene Co-expression Network Analysis (WGCNA). Besides, Interferome Database was used for predicting the transcriptional factors possessing IFN-I promoter-binding sites to the key IFN-I genes for COVID-19 diagnosis.ResultsIn this study, we identified a highly-preserved gene module between SARS-CoV-2 infected nasal swab and postmortem lung tissue regulating IFN-I signaling for COVID-19 complementary diagnosis, in which the following 14 IFN-I-stimulated genes are highly-conserved, including BST2, IFIT1, IFIT2, IFIT3, IFITM1, ISG15, MX1, MX2, OAS1, OAS2, OAS3, OASL, RSAD2, and STAT1. The stratified severity of COVID-19 may also be identified by the transcriptional level of these 14 IFN-I genes.ConclusionUsing transcriptional and computational analysis on RNA-seq data retrieved from NCBI-GEO, we identified a highly-preserved 14-gene transcriptional profile regulating IFN-I signaling in nasal swab and postmortem lung tissue infected by SARS-CoV-2. Such a conserved biosignature involved in IFN-I-related host response may be leveraged for COVID-19 diagnosis.

Transcriptional Profile Investigation Identified Aberrant CD34 Isoforms and Apela As a Hub Gene in IGH-DUX4 Subtype B-Cell Precursor Acute Lymphoblastic Leukemia

Background CD34, a transmembrane sialoglycoprotein, is expressed in hematopoietic stem and progenitor cells, endothelial cells, and bone marrow stromal cells. Therefore, in the clinical diagnosis and classification of leukemia, high expression of CD34 is customized as a marker for the immature phenotype. Although alternative splicing is a common oncogenic mechanism in various cancers, abnormal splicing of CD34 has not been revealed in hematological malignancies. Methods Here, we investigated the transcriptional profile of CD34, including expression level and alternative splicing, by RNA sequencing (RNA-seq) analysis from hematological malignancies and normal bone marrow samples. The raw sequencing reads were aligned to human reference genome hg38 using HISAT2, followed by featureCounts quantification, and co-expressed and differentially expressed genes (DEGs) were detected by WGCNA and DESeq2, respectively. Alternative splicing events were calculated by rMATS, and further validated by reverse transcriptase PCR (RT-PCR). Results The transcriptome characteristics of hematological malignancies, including AML, B-ALL, T-ALL, and MPAL, were elaborately investigated. We found that two accompanying novel in-frame splicing isoforms of CD34 were exclusively detected in B-ALL. Furthermore, we focused on B-ALL (n = 504) to systematically explore the transcriptional profile of CD34. The two novel splicing isoforms share a common first exon at the 5' untranslated region of CD34 (Figure 1B), suggesting an alternative promoter that mediated the splicing. We further observe that the novel aberrant CD34 isoforms are mainly accompanied by IGH-DUX4 gene fusion, which has been reported characterized by high expression of CD34 and intragenic ERG deletion. All IGH-DUX4 B-ALL cases (n = 20) in our cohort were positive with the novel aberrant CD34 isoforms and validated by RT-PCR (Figure 1A). We also validated the negative result for the aberrant CD34 isoforms in ZNF384-r (n = 32) B-ALL in our cohort, which is also characterized by high CD34 expression. According to the RNA-seq based pseudo-time analysis of our B-ALL cohort, IGH-DUX4 cases mostly tended to be common-B ALL, while ZNF384-r cases presented with more Pro-B ALL, which is also in accordance with the previous report based on immunophenotype analysis. To further discover the discrepancy between DUX4-r and ZNF384-r B-ALL subtypes, we compared DEGs and functional enrichment analysis between these two subtypes with B-other ALL as control, respectively (Figure 2). Activation of JAK-STAT signaling and downregulation of SOCS2, which acts as a negative controller of JAK-STAT signaling, were observed in both subtypes (Figure 2). The hematopoietic cell lineage gene set was enriched in ZNF384-r, consistent with its more immature phenotype. The co-expression gene network for IGH-DUX4 was constructed, and the hub gene APELA was emphasized among the closely related genes (Figure 3). Discussion In this study, we firstly identified two novel in-frame CD34 isoforms that share a common alternative first exon at its 5'UTR and are highly enriched in the IGH-DUX4 subtype B-ALL. It has been reported that intragenic focal ERG deletion is characteristically co-occurred with IGH-DUX4, and ERG targets CD34 through regulates its super-enhancer to affect the transcription program. It needs further investigation whether ERG deletion contributes to the aberrant transcription of CD34 in IGH-DUX4 subtype B-ALL. We also emphasized APELA as a hub gene in the transcription regulation network of IGH-DUX4. As it currently lacks effective targeted therapy for IGH-DUX4 subtype B-ALL, the potential therapeutic significance of the closely accompanied aberrant CD34 isoforms and APELA as a hub gene deserves further investigation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Role of cFOS in mechanosensitive transcriptional regulation in diabetes associated atherosclerosis

Atherosclerosis, as manifested clinically by myocardial infarction, stroke and peripheral vascular disease, is a major contributor to cardiovascular disease, the leading cause of death in patients with diabetes. The lipid-laden plaque development within the arterial vessel wall is a progressive process initiated with endothelial cell activation and monocyte adhesion. These cellular events occur primarily at the regions of blood vessels exposed to turbulent blood flow (TBF) and low shear stress such as vascular bends and bifurcations. Exposure of the vascular cells to chronic hyperglycaemia and TBF induces a proatherogenic transcriptional profile. Studies have shown that shear stress regulates vascular pathophysiology via differential regulation of transcription factors (TFs) such as KLF4, EGR1 and AP-1, hence named as mechanosensitive TFs. AP-1 is a heterodimer composed of FOS, Jun and ATF family of TFs. Studies have shown that it is activated by low shear stress in cultured endothelial cells. Increasing evidence supports the vital role of AP-1 family members in inflammation and diabetes-induced myocardial dysfunction. However, gene targets and the mechanisms underlying hyperglycemia-induced activation of AP-1 transcription factor cFOS in vascular regions exposed to TBF are not known.Although a novel approach not previously studied in diabetes associated atherosclerosis, we used a single cell RNA sequencing (scRNA-seq) approach to identify endothelial cells from TBF regions of aorta. Diabetes was induced with streptozotocin (STZ) in Apoe−/− mice and followed for 10 weeks. Cells from digested aortae of control and diabetic mice were subjected to scRNA-seq using 10X Genomics system and Illumina Nova-seq 6000. Unsupervised graph based clustering grouped cells into fourteen cell clusters with similar gene expression profile. We applied a list of mechanosensitive gene markers including EGR1, cFOS, Junb and ICAM1 in scRNA-seq analysis to identify endothelial cells from TBF regions of aorta. This approach identified atheroprone endothelial cells exposed to persistent TBF that showed a distinct transcriptional profile with more than six hundred genes differentially expressed. Importantly, cFOS was the most significantly upregulated gene in endothelial cells exposed to TBF. We next generated adiabetes associated transcriptional signature unique to endothelial cells exposed to TBF as compared to all other cell types in the aorta. We identified several genes in endothelial cells exposed to TBF and hyperglycaemia uniquely dysregulated in diabetic Apoe−/− mice as compared to control mice (cut off = FDR<0.05, fold change at least 2-fold). Gene set enrichment analysis identified “fluid shear stress and atherosclerosis” as most significantly dysregulated pathway in endothelial cells. These novel findings indicate that AP-1 TF subunit cFOS is a potential therapeutic target in diabetes associated atherosclerosis that warrant further experimental exploration. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): National Heart Foundation of Australia

Transcriptional Profile of Cytokines, Regulatory Mediators and TLR in Mesenchymal Stromal Cells after Inflammatory Signaling and Cell-Passaging

Adult human subcutaneous adipose tissue (AT) harbors a rich population of mesenchymal stromal cells (MSCs) that are of interest for tissue repair. For this purpose, it is of utmost importance to determine the response of AT-MSCs to proliferative and inflammatory signals within the damaged tissue. We have characterized the transcriptional profile of cytokines, regulatory mediators and Toll-like receptors (TLR) relevant to the response of MSCs. AT-MSCs constitutively present a distinct profile for each gene and differentially responded to inflammation and cell-passaging. Inflammation leads to an upregulation of IL-6, IL-8, IL-1β, TNFα and CCL5 cytokine expression. Inflammation and cell-passaging increased the expression of HGF, IDO1, PTGS1, PTGS2 and TGFβ. The expression of the TLR pattern was differentially modulated with TLR 1, 2, 3, 4, 9 and 10 being increased, whereas TLR 5 and 6 downregulated. Functional enrichment analysis demonstrated a complex interplay between cytokines, TLR and regulatory mediators central for tissue repair. This profiling highlights that following a combination of inflammatory and proliferative signals, the sensitivity and responsive capacity of AT-MSCs may be significantly modified. Understanding these transcriptional changes may help the development of novel therapeutic approaches.