scholarly journals Splice Factor Polypyrimidine tract-binding protein 1 (Ptbp1) is Required for Immune Priming of the Endothelium in Atherogenic Disturbed Flow Conditions

2021 ◽  
Author(s):  
Jessica A Hensel ◽  
Sarah-Anne E Nicholas ◽  
Evan R Jellison ◽  
Amy L Kimble ◽  
Antoine Menoret ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Immune Cell ◽  
Myeloid Cell ◽  
Innate Immune ◽  
Polypyrimidine Tract ◽  
Cell Recruitment ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Immune Cell Recruitment

NFkB mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through upregulation of adhesion molecules Icam1 and Vcam. The endothelium is primed for cytokine activation of NFkB by exposure to low and disturbed blood flow (LDF) in vivo and by LDF or static conditions in cultured cells. While priming leads to an exaggerated expression of Icam1 and Vcam following cytokine stimulation, the molecular underpinnings are not fully understood. We showed that alternative splicing of genes regulating NFkB signaling occurs during priming, but the functional implications of this are not known. We hypothesize that the regulation of splicing by RNA-binding splice factors is critical for priming. Here, we perform a CRISPR screen in cultured aortic endothelial cells to determine whether splice factors active in the response to LDF participate in endothelial cell priming. Using Icam1 and Vcam induction by TNFalpha stimulation as a marker of priming, we identify polypyrimidine tract binding protein (Ptbp1) as a required splice factor. Ptbp1 expression is increased and its motifs are enriched nearby alternatively spliced exons in endothelial cells exposed to LDF in vivo in a platelet dependent manner, indicating its induction by early innate immune cell recruitment. At a mechanistic level, deletion of Ptbp1 inhibited NFkB nuclear translocation and transcriptional activation. These changes coincided with altered splicing of key components of the NFkB signaling pathway that were similarly altered in the LDF response. However, these splicing and transcriptional changes could be restored by expression of human PTBP1 cDNA in Ptbp1 deleted cells. In vivo, endothelial specific deletion of Ptbp1 reduced myeloid cell infiltration at regions of LDF in atherosclerotic mice. In human coronary arteries, PTBP1 expression correlates with expression of TNF pathway genes and amount of plaque. Together, our data suggest that Ptbp1, which is activated in the endothelium by innate immune cell recruitment in regions of LDF, is required for priming of the endothelium for subsequent NFkB activation and myeloid cell recruitment in vascular inflammation.

scholarly journals Interleukin-17D and Nrf2 mediate initial innate immune cell recruitment and restrict MCMV infection

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Ruth Seelige ◽  
Robert Saddawi-Konefka ◽  
Nicholas M. Adams ◽  
Gaëlle Picarda ◽  
Joseph C. Sun ◽  
...  
Keyword(s):  
Immune Cell ◽  
Innate Immune ◽  
Innate Immune Cell ◽  
Mcmv Infection ◽  
Cell Recruitment ◽  
Immune Cell Recruitment

scholarly journals The Cutaneous Wound Innate Immunological Microenvironment

2020 ◽  
Vol 21 (22) ◽  
pp. 8748
Author(s):  
Stephen Kirchner ◽  
Vivian Lei ◽  
Amanda S. MacLeod
Keyword(s):  
Immune Response ◽  
Wound Repair ◽  
Immune Cell ◽  
Skin Barrier ◽  
Innate Immune ◽  
Immune Microenvironment ◽  
Cell Recruitment ◽  
Adaptive Processes ◽  
Immune Cell Recruitment

The skin represents the first line of defense and innate immune protection against pathogens. Skin normally provides a physical barrier to prevent infection by pathogens; however, wounds, microinjuries, and minor barrier impediments can present open avenues for invasion through the skin. Accordingly, wound repair and protection from invading pathogens are essential processes in successful skin barrier regeneration. To repair and protect wounds, skin promotes the development of a specific and complex immunological microenvironment within and surrounding the disrupted tissue. This immune microenvironment includes both innate and adaptive processes, including immune cell recruitment to the wound and secretion of extracellular factors that can act directly to promote wound closure and wound antimicrobial defense. Recent work has shown that this immune microenvironment also varies according to the specific context of the wound: the microbiome, neuroimmune signaling, environmental effects, and age play roles in altering the innate immune response to wounding. This review will focus on the role of these factors in shaping the cutaneous microenvironment and how this ultimately impacts the immune response to wounding.

scholarly journals Transient Expression of Cellular Polypyrimidine-Tract Binding Protein Stimulates Cap-Independent Translation Directed by Both Picornaviral and Flaviviral Internal Ribosome Entry Sites In Vivo

2000 ◽  
Vol 20 (5) ◽  
pp. 1583-1595 ◽  
Author(s):  
Rainer Gosert ◽  
Ki Ha Chang ◽  
Rene Rijnbrand ◽  
MinKyung Yi ◽  
David V. Sangar ◽  
...  
Keyword(s):  
Binding Protein ◽  
Fold Increase ◽  
Null Mutant ◽  
Polypyrimidine Tract ◽  
Reporter Protein ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Independent Translation

ABSTRACT The regulation of cap-independent translation directed by the internal ribosome entry sites (IRESs) present in some viral and cellular RNAs is poorly understood. Polypyrimidine-tract binding protein (PTB) binds specifically to several viral IRESs. IRES-directed translation may be reduced in cell-free systems that are depleted of PTB and restored by reconstitution of lysates with recombinant PTB. However, there are no data concerning the effects of PTB on IRES-directed translation in vivo. We transfected cells with plasmids expressing dicistronic transcripts in which the upstream cistron encoded PTB or PTB deletion mutants (including a null mutant lacking amino acid residues 87 to 531). The downstream cistron encoded a reporter protein (chloramphenicol acetyltransferase [CAT]) under translational control of the poliovirus IRES which was placed within the intercistronic space. In transfected BS-C-1 cells, transcripts expressing wild-type PTB produced 12-fold more reporter protein than similar transcripts encoding the PTB null mutant. There was a 2.4-fold difference in CAT produced from these transcripts in HeLa cells, which contain a greater natural abundance of PTB. PTB similarly stimulated CAT production from transcripts containing the IRES of hepatitis A virus or hepatitis C virus in BS-C-1 cells and Huh-7 cells (37- to 44-fold increase and 5 to 5.3-fold increase, respectively). Since PTB had no quantitative or qualitative effect on transcription from these plasmids, we conclude that PTB stimulates translation of representative picornaviral and flaviviral RNAs in vivo. This is likely to reflect the stabilization of higher ordered RNA structures within the IRES and was not observed with PTB mutants lacking RNA recognition motifs located in the C-terminal third of the molecule.

scholarly journals Polypyrimidine Tract-Binding Protein Positively Regulates Inclusion of an Alternative 3′-Terminal Exon

1999 ◽  
Vol 19 (1) ◽  
pp. 78-85 ◽  
Author(s):  
Hua Lou ◽  
David M. Helfman ◽  
Robert F. Gagel ◽  
Susan M. Berget
Keyword(s):  
Binding Protein ◽  
Consensus Sequence ◽  
Polypyrimidine Tract ◽  
Terminal Exon ◽  
Exon Inclusion ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Exon 4 ◽  
A Site

ABSTRACT Polypyrimidine tract-binding protein (PTB) is an abundant vertebrate hnRNP protein. PTB binding sites have been found within introns both upstream and downstream of alternative exons in a number of genes that are negatively controlled by the binding of PTB. We have previously reported that PTB binds to a pyrimidine tract within an RNA processing enhancer located adjacent to an alternative 3′-terminal exon within the gene coding for calcitonin and calcitonin gene-related peptide. The enhancer consists of a pyrimidine tract and CAG directly abutting on a 5′ splice site sequence to form a pseudoexon. Here we show that the binding of PTB to the enhancer pyrimidine tract is functional in that exon inclusion increases when in vivo levels of PTB increase. This is the first example of positive regulation of exon inclusion by PTB. The binding of PTB was antagonistic to the binding of U2AF to the enhancer-located pyrimidine tract. Altering the enhancer pyrimidine tract to a consensus sequence for the binding of U2AF eliminated enhancement of exon inclusion in vivo and exon polyadenylation in vitro. An additional PTB binding site was identified close to the AAUAAA hexanucleotide sequence of the exon 4 poly(A) site. These observations suggest a dual role for PTB in facilitating recognition of exon 4: binding to the enhancer pyrimidine tract to interrupt productive recognition of the enhancer pseudoexon by splicing factors and interacting with the poly(A) site to positively affect polyadenylation.

scholarly journals Differentiation-induced Colocalization of the KH-type Splicing Regulatory Protein with Polypyrimidine Tract Binding Protein and the c-srcPre-mRNA

2004 ◽  
Vol 15 (2) ◽  
pp. 774-786 ◽  
Author(s):  
Megan P. Hall ◽  
Sui Huang ◽  
Douglas L. Black
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Neuroblastoma Cell ◽  
Regulatory Protein ◽  
Neuroblastoma Cells ◽  
Neuroblastoma Cell Line ◽  
Polypyrimidine Tract ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein

We have examined the subcellular localization of the KH-type splicing regulatory protein (KSRP). KSRP is a multidomain RNA-binding protein implicated in a variety of cellular processes, including splicing in the nucleus and mRNA localization in the cytoplasm. We find that KSRP is primarily nuclear with a localization pattern that most closely resembles that of polypyrimidine tract binding protein (PTB). Colocalization experiments of KSRP with PTB in a mouse neuroblastoma cell line determined that both proteins are present in the perinucleolar compartment (PNC), as well as in other nuclear enrichments. In contrast, HeLa cells do not show prominent KSRP staining in the PNC, even though PTB labeling identified the PNC in these cells. Because both PTB and KSRP interact with the c-src transcript to affect N1 exon splicing, we examined the localization of the c-src pre-mRNA by fluorescence in situ hybridization. The src transcript is present in specific foci within the nucleus that are presumably sites of src transcription but are not generally perinucleolar. In normally cultured neuroblastoma cells, these src RNA foci contain PTB, but little KSRP. However, upon induced neuronal differentiation of these cells, KSRP occurs in the same foci with src RNA. PTB localization remains unaffected. This differentiation-induced localization of KSRP with src RNA correlates with an increase in src exon N1 inclusion. These results indicate that PTB and KSRP do indeed interact with the c-src transcript in vivo, and that these associations change with the differentiated state of the cell.

scholarly journals PARG Suppresses Tumorigenesis and Downregulates Genes Controlling Angiogenesis, Inflammatory Response, and Immune Cell Recruitment

2022 ◽  
Author(s):  
Sarah Johnson ◽  
Yaroslava Karpova ◽  
Danping Guo ◽  
Atreyi Ghatak ◽  
Dmitriy A. Markov ◽  
...  
Keyword(s):  
Immune Cell ◽  
Critical Role ◽  
Tumor Growth Inhibition ◽  
Rna Seq ◽  
Cell Recruitment ◽  
Tumor Onset ◽  
Tumor Tissues ◽  
Immune Cell Recruitment

Abstract Chemokines are highly expressed in tumor microenvironment and play a critical role in all aspects of tumorigenesis, including the recruitment of tumor-promoting immune cells, activation of cancer-associated fibroblasts, angiogenesis, metastasis, and growth. Poly(ADP-ribose) polymerase (PARP) is a multi-target transcription regulator with high levels of poly(ADP-ribose) (pADPr) being reported in a variety of cancers. Furthermore, poly(ADP-ribose) glycohydrolase (PARG), an enzyme that degrades pADPr, has been reported to be downregulated in tumor tissues with abnormally high levels of pADPr. In conjunction to this, we have recently reported that the reduction of pADPr, by either pharmacological inhibition of PARP or PARG’s overexpression, disrupts renal carcinoma cell malignancy in vitro. Here, we use 3T3 mouse embryonic fibroblasts, a universal model for malignant transformation, to follow the effect of PARG upregulation on cells’ tumorigenicity in vivo. We found that the overexpression of PARG in mouse allografts produces significantly smaller tumors with a delay in tumor onset. As downregulation of PARG has also been implicated in promoting the activation of pro-inflammatory genes, we also followed the gene expression profile of PARG-overexpressing 3T3 cells using RNA-seq approach and observed that chemokine transcripts are significantly reduced in those cells. Our data suggest that the upregulation of PARG may be potentially useful for the tumor growth inhibition in cancer treatment and as anti-inflammatory intervention.

scholarly journals Effects of VEGF blockade on the dynamics of the inflammatory landscape in glioblastoma-bearing mice

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Aurélie Soubéran ◽  
Sophie Brustlein ◽  
Caroline Gouarné ◽  
Lionel Chasson ◽  
Aurélie Tchoghandjian ◽  
...  
Keyword(s):  
Mouse Model ◽  
Immune Cell ◽  
Early Stage ◽  
Basal Area ◽  
Myeloid Cell ◽  
Innate Immune ◽  
Therapeutic Modality ◽  
Detectable Effect ◽  
Anti Vegf

Abstract Background Targeting angiogenesis has been and continues to be an attractive therapeutic modality in glioblastoma (GBM) patients. However, GBM rapidly becomes refractory to anti-VEGF therapies. Myeloid cell infiltration is an important determinant of tumor progression. Given that VEGF is a modulator of the innate immune response we sought to analyze the dynamics of this response in a mouse model of GBM undergoing anti-VEGF therapy. Methods We grafted GL261-DsRed cells in transgenic Thy1-CFP//LysM-EGFP//CD11c-EYFP reporter mice. We combined recurrent spectral two-photon imaging with multiparametric cytometry, immunostaining, and brain clearing to characterize at two critical stages of tumor development (day 21 and day 28 after tumor grafting) the nature and spatial distribution of the innate response in control and bevacizumab-treated mice. Results We report that at an early stage (21 day), VEGF blockade has a detectable effect on the number of microglial cells but only a mild effect on the number of infiltrating myeloid cells. At a later stage (day 28), the treatment resulted in a specific adjustment of dendritic cell subsets. In treated mice, the number of monocytes and their monocyte-derived dendritic cells (moDC) progeny was increased by approximately twofold compared to untreated mice. In agreement, by in vivo quantitative imaging, we observed that treatment increased the number of LysM-EGFP cells traveling in tumor blood vessels and doubled the densities of both infiltrated LysM-EGFP monocytes and double-labeled EGFP/EYFP moDC. The treatment also led to an increased density of conventional cDCs2 subset together with a decrease of cDCs1 subset, necessary for the development of anti-tumor immunity. Finally, we describe differential spatial cell distributions and two immune cell-traveling routes into the brain. LysM-EGFP cells distributed as a gradient from the meninges towards the tumor whereas CD11c-EYFP/MHCII+ cells were located in the basal area of the tumor. Brain clearing also revealed a flow of CD11c-EYFP cells following the corpus callosum. Conclusion We uncovered new features in the dynamics of innate immune cells in GBM-bearing mice and deciphered precisely the key populations, i.e., DC subsets controlling immune responses, that are affected by VEGF blockade. Since despite differences, human pathogenesis presents similarities with our mouse model, the data provide new insights into the effect of bevacizumab at the cellular level.

Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation

Science ◽  
2019 ◽  
Vol 363 (6425) ◽  
pp. eaat7554 ◽  
Author(s):  
Marta Joana Costa Jordão ◽  
Roman Sankowski ◽  
Stefanie M. Brendecke ◽  
Sagar ◽  
Giuseppe Locatelli ◽  
...  
Keyword(s):  
Single Cell ◽  
Immune Cell ◽  
Myeloid Cell ◽  
Innate Immune ◽  
Functional Studies ◽  
Cell Diversity ◽  
Single Cell Profiling ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

The innate immune cell compartment is highly diverse in the healthy central nervous system (CNS), including parenchymal and non-parenchymal macrophages. However, this complexity is increased in inflammatory settings by the recruitment of circulating myeloid cells. It is unclear which disease-specific myeloid subsets exist and what their transcriptional profiles and dynamics during CNS pathology are. Combining deep single-cell transcriptome analysis, fate mapping, in vivo imaging, clonal analysis, and transgenic mouse lines, we comprehensively characterized unappreciated myeloid subsets in several CNS compartments during neuroinflammation. During inflammation, CNS macrophage subsets undergo self-renewal, and random proliferation shifts toward clonal expansion. Last, functional studies demonstrated that endogenous CNS tissue macrophages are redundant for antigen presentation. Our results highlight myeloid cell diversity and provide insights into the brain’s innate immune system.

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