Focus Forming Virus Proviral Integration Research Articles

Cigarette Smoke Extract-Treated Mouse Airway Epithelial Cells-Derived Exosomal LncRNA MEG3 Promotes M1 Macrophage Polarization and Pyroptosis in Chronic Obstructive Pulmonary Disease by Upregulating TREM-1 via m6A Methylation.

Cigarette smoke extract (CSE)-treated mouse airway epithelial cells (MAECs)-derived exosomes accelerate the progression of chronic obstructive pulmonary disease (COPD) by upregulating triggering receptor expressed on myeloid cells 1 (TREM-1); however, the specific mechanism remains unclear. We aimed to explore the potential mechanisms of CSE-treated MAECs-derived exosomes on M1 macrophage polarization and pyroptosis in COPD. In vitro, exosomes were extracted from CSE-treated MAECs, followed by co-culture with macrophages. In vivo, mice exposed to cigarette smoke (CS) to induce COPD, followed by injection or/and intranasal instillation with oe-TREM-1 lentivirus. Lung function and pathological changes were evaluated. CD68+ cell number and the levels of iNOS, TNF-α, IL-1β (M1 macrophage marker), and pyroptosis-related proteins (NOD-like receptor family pyrin domain containing 3, apoptosis-associated speck-like protein containing a caspase-1 recruitment domain, caspase-1, cleaved-caspase-1, gasdermin D [GSDMD], and GSDMD-N) were examined. The expression of maternally expressed gene 3 (MEG3), spleen focus forming virus proviral integration oncogene (SPI1), methyltransferase 3 (METTL3), and TREM-1 was detected and the binding relationships among them were verified. MEG3 increased N6-methyladenosine methylation of TREM-1 by recruiting SPI1 to activate METTL3. Overexpression of TREM-1 or METTL3 negated the alleviative effects of MEG3 inhibition on M1 polarization and pyroptosis. In mice exposed to CS, EXO-CSE further aggravated lung injury, M1 polarization, and pyroptosis, which were reversed by MEG3 inhibition. TREM-1 overexpression negated the palliative effects of MEG3 inhibition on COPD mouse lung injury. Collectively, CSE-treated MAECs-derived exosomal long non-coding RNA MEG3 may expedite M1 macrophage polarization and pyroptosis in COPD via the SPI1/METTL3/TREM-1 axis.

Interferon regulatory factor 2 binding protein 2b regulates neutrophil versus macrophage fate during zebrafish definitive myelopoiesis.

Aproper choice of neutrophil-macrophage progenitor cell fate is essential for the generation of adequate myeloid subpopulations during embryonic development and in adulthood. The network governing neutrophil-macrophage progenitor cell fate has several key determinants, such as myeloid master regulators CCAAT enhancer binding protein alpha (C/EBPα) and spleen focus forming virus proviral integration oncogene (PU.1). Nevertheless, more regulators remain to be identified and characterized. To ensure balanced commitment of neutrophil-macrophage progenitors toward each lineage, the interplay among these determinants is not only synergistic, but also antagonistic. Depletion of interferon regulatory factor 2 binding protein 2b (Irf2bp2b), a well-known negative transcription regulator, results in a bias in neutrophil-macrophage progenitor cell fate in favor of macrophages at the expense of neutrophils during the stage of definitive myelopoiesis in zebrafish embryos. Mechanistic studies indicate that Irf2bp2b acts as a downstream target of C/EBPα, repressing PU.1 expression, and that SUMOylation confers the repressive function of Irf2bp2b. Thus, Irf2bp2b is a novel determinant in the choice of fate of neutrophil-macrophage progenitor cells.

Identification of key transcription factors associated with cerebral ischemia‑reperfusion injury based on gene‑set enrichment analysis.

Cerebral ischemia-reperfusion injury (CIRI) usually causes detrimental complications following reperfusion therapy in stroke patients. The present study systematically investigated the regulatory mechanism involved in the pathogenesis of CIRI using gene set enrichment analysis of the transient middle cerebral artery occlusion mouse stroke model. The results revealed a total of 13 CIRI-related transcription factors (TFs), including CCAAT enhancer binding protein b (Cebpb), Cebpa, early growth response-1, Fos, Rela, Jund, signal transduction and activator of transcription 5a/b, transformation related protein 53, GLI family zinc finger 2 (Gli2), Sp3, TF AP-2 α (Tfap2a) and spleen focus forming virus proviral integration oncogene (Spi1). To the best of our knowledge, five TFs (Cebpa, Gli2, Sp3, Tfap2a and Spi1) were the first to be reported associated with CIRI in the present study. The five novel CIRI-related TFs were mainly associated with pathways of inflammation and responses to reperfusion, including the tumor necrosis factor signaling pathway (Gli2, Spi1 and Tfap2a, P=0.0035, 0.0035 and 0.048, respectively), interleuking-17 signaling pathway (Cebpa, Gli2, Sp3, Spi1 and Tfap2a, P=0.019, 0.047, 0.019, 0.035 and 0.005, respectively) and fluid shear stress and atherosclerosis (Gli2, Sp3, Spi1 and Tfap2a, P=0.047, 0.046, 0.013 and 0.003, respectively). These results may improve understanding of the potential molecular mechanism underlying the pathogenesis of CIRI at the genome-wide level.

Distinct regulatory networks control the development of macrophages of different origins in zebrafish

AbstractMacrophages are key components of the innate immune system and play pivotal roles in immune response, organ development, and tissue homeostasis. Studies in mice and zebrafish have shown that tissue-resident macrophages derived from different hematopoietic origins manifest distinct developmental kinetics and colonization potential, yet the genetic programs controlling the development of macrophages of different origins remain incompletely defined. In this study, we use zebrafish, where tissue-resident macrophages arise from the rostral blood island (RBI) and ventral wall of dorsal aorta (VDA), the zebrafish hematopoietic tissue equivalents to the mouse yolk sac and aorta-gonad-mesonephros for myelopoiesis, to address this issue. We show that RBI- and VDA-born macrophages are orchestrated by distinctive regulatory networks formed by the E-twenty-six (Ets) transcription factors Pu.1 and Spi-b, the zebrafish ortholog of mouse spleen focus forming virus proviral integration oncogene B (SPI-B), and the helix-turn-helix DNA-binding domain containing protein Irf8. Epistatic studies document that during RBI macrophage development, Pu.1 acts upstream of Spi-b, which, upon induction by Pu.1, partially compensates the function of Pu.1. In contrast, Pu.1 and Spi-b act in parallel and cooperatively to regulate the development of VDA-derived macrophages. Interestingly, these two distinct regulatory networks orchestrate the RBI- and VDA-born macrophage development largely by regulating a common downstream gene, Irf8. Our study indicates that macrophages derived from different origins are governed by distinct genetic networks formed by the same repertoire of myeloid-specific transcription factors.

Retinoic acid triggers c-kit gene expression in spermatogonial stem cells through an enhanceosome constituted between transcription factor binding sites for retinoic acid response element (RARE), spleen focus forming virus proviral integration oncogene (SPFI1) (PU.1) and E26 transformation-specific (ETS).

Restricted availability of retinoic acid (RA) in the testicular milieu regulates transcriptional activity of c-kit (KIT, CD117), which aids in the determination of spermatogonial stem-cell differentiation. The effect of RA on c-kit has been reported previously, but its mode of genomic action remains unresolved. We studied the molecular machinery guiding RA responsiveness to the c-kit gene using spermatogonial stem-cell line C18-4 and primary spermatogonial cells. A novel retinoic acid response element (RARE) positioned at -989 nucleotides upstream of the transcription start site (TSS) was identified, providing a binding site for a dimeric RA receptor (i.e. retinoic acid receptor gamma (RARγ) and retinoic X receptor). RA treatment influenced c-kit promoter activity, along with endogenous c-kit expression in C18-4 cells. A comprehensive promoter deletion assay using the pGL3B reporter system characterised the region spanning -271bp and -1011bp upstream of the TSS, which function as minimal promoter and maximal promoter, respectively. In silico analysis predicted that the region -1011 to +58bp comprised the distal enhancer RARE and activators such as spleen focus forming virus proviral integration oncogene (SPFI1) (PU.1), specificity protein 1 (SP1) and four E26 transformation-specific (ETS) tandem binding sites at the proximal region. Gel retardation and chromatin immunoprecipitation (ChIP) assays showed binding for RARγ, PU.1 and SP1 to the predicted consensus binding sequences, whereas GABPα occupied only two out of four ETS binding sites within the c-kit promoter region. We propose that for RA response, an enhanceosome is orchestrated through scaffolding of a CREB-binding protein (CBP)/p300 molecule between RARE and elements in the proximal promoter region, controlling germ-line expression of the c-kit gene. This study outlines the fundamental role played by RARγ, along with other non-RAR transcription factors (PU.1, SP1 and GABPα), in the regulation of c-kit expression in spermatogonial stem cells in response to RA.

RNA Profiling in Human and Murine Transplanted Hearts: Identification and Validation of Therapeutic Targets for Acute Cardiac and Renal Allograft Rejection

Acute cellular rejection (ACR) is the adverse response of the recipient's immune system against the allogeneic graft. Using human surveillance endomyocardial biopsies (EMBs) manifesting ACR and murine allogeneic grafts, we profiled implicated microRNAs (miRs) and mRNAs. MiR profiling showed that miR‐21, ‐142‐3p, ‐142‐5p, ‐146a, ‐146b, ‐155, ‐222, ‐223, and ‐494 increased during ACR in humans and mice, whereas miR‐149‐5p decreased. mRNA profiling revealed 70 common differentially regulated transcripts, all involved in immune signaling and immune‐related diseases. Interestingly, 33 of 70 transcripts function downstream of IL‐6 and its transcription factor spleen focus forming virus proviral integration oncogene (SPI1), an established target of miR‐155, the most upregulated miR in human EMBs manifesting rejection. In a mouse model of cardiac transplantation, miR‐155 absence and pharmacological inhibition attenuated ACR, demonstrating the causal involvement and therapeutic potential of miRs. Finally, we corroborated our miR signature in acute cellular renal allograft rejection, suggesting a nonorgan specific signature of acute rejection. We concluded that miR and mRNA profiling in human and murine ACR revealed the shared significant dysregulation of immune genes. Inflammatory miRs, for example miR‐155, and transcripts, in particular those related to the IL‐6 pathway, are promising therapeutic targets to prevent acute allograft rejection.

Transcription factor regulatory network for early lung immune response to tuberculosis in mice.

Numerous transcription factors (TFs) have been suggested to have a role in Mycobacterium tuberculosis infection; however, the TFs involved in the early immune response of lung cells remains to be fully elucidated. The present study aimed to identify TFs which may have a role in the early immune response to tuberculosis and the gene regulatory networks in which they are involved. Gene expression data obtained from microarray analysis of the early lung immune response to tuberculosis (Gene Expression Omnibus; accession no. GSE23014) was integrated with data for TF binding sites and protein-protein interactions in order to construct a TF regulatory network. The role of TFs in protein complexes, active modules, topology of the network and regulation of immune processes were investigated. The results demonstrated that the constructed gene regulatory network harbored 1,270 differentially expressed (DE) genes with 4,070 regulatory and protein-protein interactions. In addition, it was revealed that 17 DE TFs were involved in the positive regulation of numerous immunological and biological processes, including T cell activation, T cell proliferation and tuberculosis-associated gene expression, in the constructed regulatory network. Signal transducer and activator of transcription 4, interferon regulatory factor 8, spleen focus-forming virus proviral integration 1, enhancer of zeste homolog 2 and kruppel-like factor 4 were predicted to be the primary TFs regulating the DE genes during early lung infection by M. tuberculosis, as determined through various analyses of the gene regulatory network. In conclusion, the present study identified novel TFs involved in the early response to M. tuberculosis infection, which may enhance current understanding of the molecular mechanism underlying tuberculosis infection and introduced potential targets for novel tuberculosis therapies.

A myelopoiesis gene signature during remission in anti-neutrophil cytoplasm antibody-associated vasculitis does not predict relapses but seems to reflect ongoing prednisolone therapy

A myelopoiesis gene signature in circulating leucocytes, exemplified by increased myeloperoxidase (MPO) and proteinase 3 (PR3) mRNA levels, has been reported in patients with active anti-neutrophil cytoplasm antibody-associated vasculitis (AAV), and to a lesser extent during remission. We hypothesized that this signature could predict disease relapse. mRNA levels of PR3, MPO, selected myelopoiesis transcription factors [CCAAT/enhancer binding protein α (CEBP-α), CCAAT/enhancer binding protein β (CEBP-β), SPI1/PU.1-related transcription factor (SPIB), spleen focus forming virus proviral integration oncogene, PU.1 homologue (SPI1)] and microRNAs (miRNAs) from patient and control peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMN) were analysed and associated with clinical data. Patients in stable remission had higher mRNA levels for PR3 (PBMC, PMN) and MPO (PBMC). PR3 and SPIB mRNA correlated positively in controls but negatively in patient PBMC. Statistically significant correlations existed between PR3 mRNA and several miRNAs in controls, but not in patients. PR3/MPO mRNA levels were not associated with previous or future relapses, but correlated with steroid treatment. Prednisolone doses were negatively linked to SPIB and miR-155-5p, miR-339-5p (PBMC) and to miR-221, miR-361 and miR-505 (PMN). PR3 mRNA in PBMC correlated with time since last flare, blood leucocyte count and estimated glomerular filtration rate. Our results show that elevated leucocyte PR3 mRNA levels in AAV patients in remission do not predict relapse. The origin seems multi-factorial, but to an important extent explainable by prednisolone action. Gene signatures in patients with AAV undergoing steroid treatment should therefore be interpreted accordingly.

The Transcription Factor PU.1 Regulates γδ T Cell Homeostasis

BackgroundT cell development results in the generation of both mature αβ and γδ T cells. While αβ T cells predominate in secondary lymphoid organs, γδ T cells are more abundant in mucosal tissues. PU.1, an Ets family transcription factor, also identified as the spleen focus forming virus proviral integration site-1 (Sfpi1) is essential for early stages of T cell development, but is down regulated during the DN T-cell stage.Methodology/Principal FindingsIn this study, we show that in mice specifically lacking PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpi1 lck−/−) there are increased numbers of γδ T cells in spleen, thymus and in the intestine when compared to wild-type mice. The increase in γδ T cell numbers in PU.1-deficient mice is consistent in γδ T cell subsets identified by TCR variable regions. PU.1-deficient γδ T cells demonstrate greater proliferation in vivo and in vitro.Conclusions/SignificanceThe increase of γδ T cell numbers in Lck-Cre deleter strains, where deletion occurs after PU.1 expression is diminished, as well as the observation that PU.1-deficient γδ T cells have greater proliferative responses than wild type cells, suggests that PU.1 effects are not developmental but rather at the level of homeostasis. Thus, our data shows that PU.1 has a negative influence on γδ T cell expansion.

Expanded γδ T cell populations in absence of transcription factor PU.1 (36.49)

Abstract T cell development is characterized by a succession of developmental stages that lead to the generation of mature αβ and γδ T cells. γδ T cells are considered innate cells that critically contribute to host defense. PU.1, an Ets family transcription factor, also identified as the spleen focus forming virus proviral integration site-1 (Sfpi-1) is essential for early stages of T cell development and is down regulated during the pro T-cell stage. PU.1 is expressed in Th2 cells but not Th1 cells. Expression of PU.1 in other T cell subsets has not been studied extensively. In this study, we show that PU.1 is expressed in γδ T cells, and in mice that specifically lack PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpilck-/-) there are increased numbers of γδ T cells in spleen, thymus and in the intestine when compared to wild type mice. The increase in γδ T cell numbers in PU.1 deficient mice is reflected in several subsets examined (Vγ2T, Vγ3T and Vδ4T). In mice where PU.1 was deleted using a CD4-Cre transgene, the expansion of γδ T cell numbers was restricted to the intestinal population. PU.1-deficient γδ T cells secrete higher levels of IL-17 than wild type cells, but similar levels of IFN-γ. Thus, our data show that PU.1 is expressed in γδ T cells and that deletion of PU.1 results in γδ T cell expansion. This suggests that PU.1 has a negative influence on γδ T cell development or expansion.