The proprotein convertase enzyme FURIN promotes the proteolytic maturation of pro-proteins and thereby it serves as an important factor for maintaining cellular homeostasis. In T cells, FURIN is ...critical for maintaining the T regulatory cell dependent peripheral immune tolerance and intact T helper cell polarization. The enzymatic activity of FURIN is directly associated with its expression levels, but genetic determinants for cell-type specific
gene regulation have remained elusive. By exploring the histone acetyltransferase p300 binding patterns in T helper cells, a putative regulatory region at ca. 20kB upstream of
gene was identified. When this region was deleted with CRISPR/Cas9 the production of
mRNA was significantly reduced in activated mouse T cells. Genome-wide RNA profiling by sequencing revealed that the novel
regulator region also impacted the expression of several genes that have previously been associated with the Th1 type hall mark cytokine IFNγ regulation or function. Finally,
genetic regulatory region was found to specifically promote the secretion of IFNγ by activated T cells. In sum, our data unravels the presence of
expression regulatory region in T cells that has characteristics of a super-enhancer for Th1 cell fate.
Members of the substilisin/kexin like proprotein convertase (PCSK) protease family cleave and convert immature
pro-proteins into their biologically active forms. By cleaving for example prohormones, ...cytokines and cell membrane
proteins, PCSKs participate in maintaining the homeostasis in a healthy human body. Conversely, erratic enzymatic function
is thought to contribute to the pathogenesis of a wide variety of diseases, including obesity and hypercholestrolemia.
The first characterized seven PCSK enzymes (PCSK1-2, FURIN, PCSK4-7) process their substrates at a motif made up of
paired basic amino acid residues. This feature results in a variable degree of biochemical redundancy in vitro, and consequently,
shared substrate molecules between the different PCSK enzymes. This redundancy has confounded our understanding
of the specific biological functions of PCSKs. The physiological roles of these enzymes have been best illustrated
by the phenotypes of genetically engineered mice and patients that carry mutations in the PCSK genes. Recent developments
in genome-wide methodology have generated a large amount of novel information on the genetics of the first
seven proprotein convertases. In this review we summarize the reported genetic alterations and their associated phenotypes.
Thymic stromal lymphopoietin (TSLP) and IL-7 are related cytokines that mediate growth and differentiation events in the immune system. They signal through IL-7Rα-containing receptors. Target cells ...of TSLP in Th2 responses include CD4 T cells and dendritic cells (DCs). Although it has been reported that expression of TSLP receptor (TSLPR) on CD4 T cells is required for OVA-induced lung inflammation, DCs have also been shown to be target cells of TSLP. In this study, we show that murine ex vivo splenic DCs are unresponsive to TSLP, as they fail to phosphorylate STAT5, but in vitro overnight culture, especially in presence of IL-4, renders DCs responsive to both TSLP and IL-7. This induced responsiveness is accompanied by dramatic upregulation of IL-7Rα on DCs with little change in expression of TSLPR or of γ
In splenic DCs, the induction of IL-7Rα occurs mainly in CD8
DCs. In vivo, we found that IL-4 has a differential regulatory role on expression of IL-7Rα depending on the cell type; IL-4 decreases IL-7Rα expression on CD4 T cells whereas it upregulates the expression on DCs. Our results indicate that the induction of IL-7Rα expression on DCs is critical for TSLP responsiveness and that IL-4 can upregulate IL-7Rα on DCs.
Deficits in protein synthesis are associated with Parkinson's disease (PD). However, it is not known which proteins are affected or if there are synthesis differences between patients with sporadic ...and Leucine-Rich Repeat Kinase 2 (LRRK2) G2019S PD, the most common monogenic form. Here we used bio-orthogonal non-canonical amino acid tagging for global analysis of newly translated proteins in fibroblasts from sporadic and LRKK2-G2019S patients. Quantitative proteomic analysis revealed that several nascent proteins were reduced in PD samples compared to healthy without any significant change in mRNA levels. Using targeted proteomics, we validated which of these proteins remained dysregulated at the static proteome level and found that regulators of endo-lysosomal sorting, mRNA processing and components of the translation machinery remained low. These proteins included autophagy-related protein 9A (ATG9A) and translational stability regulator YTH N6-ethyladenosine RNA binding protein 3 (YTHDF3). Notably, 77% of the affected proteins in sporadic patients were also repressed in LRRK2-G2019S patients (False discovery rate (FDR) < 0.05) in both sporadic and LRRK2-G2019S samples. This analysis of nascent proteomes from PD patient skin cells reveals that regulators of proteostasis are repressed in both sporadic and LRRK2-G2019S PD.
The proprotein convertase subtilisin/kexin enzymes proteolytically convert immature proproteins into bioactive molecules, and thereby they serve as key regulators of cellular homeostasis. The ...archetype proprotein convertase subtilisin/kexin, FURIN, is a direct target gene of the IL-12/STAT4 pathway and it is upregulated in Th1 cells. We have previously demonstrated that FURIN expression in T cells critically regulates the maintenance of peripheral immune tolerance and the functional maturation of pro-TGF-β1 in vivo, but FURIN's role in cell-mediated immunity and Th polarization has remained elusive. In this article, we show that T cell-expressed FURIN is essential for host resistance against a prototypic Th1 pathogen, Toxoplasma gondii, and for the generation of pathogen-specific Th1 lymphocytes, including Th1-IL-10 cells. FURIN-deficient Th cells instead show elevated expression of IL-4R subunit α on cell surface, sensitized IL-4/STAT6 signaling, and a propensity to polarize toward the Th2 phenotype. By exploring FURIN-interacting proteins in Jurkat T cells with Strep-Tag purification and mass spectrometry, we further identify an association with a cytoskeleton modifying Ras-related C3 botulinum toxin substrate/dedicator of cytokinesis 2 protein complex and unravel that FURIN promotes F-actin polymerization, which has previously been shown to downregulate IL-4R subunit α cell surface expression and promote Th1 responses. In conclusion, our results demonstrate that in addition to peripheral immune tolerance, T cell-expressed FURIN is also a central regulator of cell-mediated immunity and Th1/2 cell balance.
•In neutrophils, IL-4 is more potent inducer of Stat6 activation than IL-13.•IFN-γ and IL-4/IL-13 regulate distinct gene expression in neutrophils.•IL-4/IL-13 induced genes are linked to MHCII ...biosynthesis oxygen or LPS responses.•IFN-γ-induced genes are related to intracellular infections.•Subjecting neutrophils to IL-4 decreases their anaerobic glycolysis.
Interleukin (IL)-4 and IL-13 are related cytokines with well-known specific roles in type 2 immune response. However, their effects on neutrophils are not completely understood. For this, we studied human primary neutrophil responses to IL-4 and IL-13. Neutrophils are dose-dependently responsive to both IL-4 and IL-13 as indicated by signal transducer and activator of transcription 6 (STAT6) phosphorylation upon stimulation, with IL-4 being more potent inducer of STAT6. IL-4-, IL-13- and Interferon (IFN)-γ-stimulated gene expression in highly purified human neutrophils induced both overlapping and unique gene expression in highly purified human neutrophils. IL-4 and IL-13 specifically regulate several immune-related genes, including IL-10, tumor necrosis factor (TNF) and leukemia inhibitory factor (LIF), while type1 immune response-related IFN-γ induced gene expression related for example, to intracellular infections. In analysis of neutrophil metabolic responses, oxygen independent glycolysis was specifically regulated by IL-4, but not by IL-13 or IFN-γ, suggesting specific role for type I IL-4 receptor in this process. Our results provide a comprehensive analysis of IL-4, IL-13 and IFN-γ -induced gene expression in neutrophils while also addressing cytokine-mediated metabolic changes in neutrophils.
TCR‐regulated protease FURIN plays an important role in T cell activation and can specifically modulate TCR‐activated transactivation.
Antigen emergence rapidly stimulates T cells, which leads to ...changes in cytokine production, cell proliferation, and differentiation. Some of the key molecules involved in these events, such as TGF‐β1 and NOTCH1, are synthesized initially as inactive precursors and are proteolytically activated during T cell activation. PCSKs regulate proprotein maturation by catalyzing the proteolytic cleavage of their substrates. The prototype PCSK FURIN is induced upon TCR activation, and its expression in T cells is critical for the maintenance of peripheral immune tolerance. In this study, we tested the hypothesis that FURIN regulates T cell activation. Our data demonstrate that IL‐2 is increased initially in FURIN‐deficient mouse CD4+ T cells, but the TCR‐induced IL‐2 mRNA expression is not sustained in the absence of FURIN. Accordingly, the inhibition of FURIN in human Jurkat T cell lines also results in a decrease in IL‐2 production, whereas the overexpression of WT FURIN is associated with elevated IL‐2 levels. In Jurkat cells, FURIN is dispensable for immediate TCR signaling steps, such as ERK, ZAP70, or LAT phosphorylation. However, with the use of gene reporter assays, we demonstrate that FURIN regulates the AP‐1, NFAT, and NF‐κB transcription factors. Finally, by performing a transcription factor‐binding site enrichment analysis on FURIN‐dependent transcriptomes, we identify the FURIN‐regulated transcription factors in mouse CD4+ T cell subsets. Collectively, our work confirms the hypothesis that the TCR‐regulated protease FURIN plays an important role in T cell activation and that it can specifically modulate TCR‐activated transactivation.
The proprotein convertase subtilisin/kexins (PCSKs) regulate biological actions by cleaving immature substrate proteins. The archetype PCSK, FURIN, promotes the pathogenicity of viruses by ...proteolytically processing viral proteins. FURIN has also important regulatory functions in both innate and adaptive immune responses but its role in the CD8+ CTLs remains enigmatic. We used a T‐cell‐specific FURIN deletion in vivo to demonstrate that FURIN promotes host response against the CTL‐dependent lymphocytic choriomeningitis virus by virtue of restricting viral burden and augmenting interferon gamma (IFNG) production. We also characterized Furin KO CD8+ T cells ex vivo, including after their activation with FURIN regulating cytokines IL12 or TGFB1. Furin KO CD8+ T cells show an inherently activated phenotype characterized by the upregulation of effector genes and increased frequencies of CD44+, TNF+, and IFNG+ cells. In the activated CTLs, FURIN regulates the productions of IL2, TNF, and GZMB and the genes associated with the TGFBR‐signaling pathway. FURIN also controls the expression of Eomes, Foxo1, and Bcl6 and the levels of ITGAE and CD62L, which implies a role in the development of CTL memory. Collectively, our data suggest that the T‐cell expressed FURIN is important for host responses in viral infections, CTL homeostasis/activation, and memory development.
We demonstrate that T‐cell‐expressed FURIN is required for normal host response against the CTL‐dependent lymphocytic choriomeningitis virus (LCMV) infection. Additionally, FURIN is important in controlling the CD8+ T‐cell homeostasis/activation and memory cell development. Overall, our data suggests that FURIN regulates the biology of CD8+ T cells.
Thymic stromal lymphopoietin (TSLP) and interleukin (IL)-7 are related cytokines that mediate growth and differentiation events in the immune system. They signal through IL-7Rα-containing receptors. ...Target cells of TSLP in Th2 responses include CD4 T cells and dendritic cells (DC's). While it has been reported that expression of TSLPR on CD4 T cells is required for OVA-induced lung inflammation, DC's have also been shown to be target cells of TSLP. We show here that murine
ex vivo
splenic DC's are unresponsive to TSLP, as they fail to phosphorylate STAT5, but
in vitro
overnight culture especially in presence of IL-4 renders DC's responsive to both TSLP and IL-7. This induced responsiveness is accompanied by dramatic upregulation of IL-7Rα on DC's with little change in expression of TSLPR or of γc. In splenic DC's, the induction of IL-7Rα occurs mainly in CD8 negative DC's.
In vivo,
we found that IL-4 has differential regulatory role on expression of IL-7Rα depending on the cell type; IL-4 decreases IL-7Rα expression on CD4 T cells while upregulating the expression on DC's. Our results indicate that the induction of IL-7Rα expression on DC's is critical for TSLP responsiveness and that IL-4 can upregulate IL-7Rα on DC's.
Introduction
R‐Ras GTPase has recently been implicated in the regulation of immune functions, particularly in dendritic cell (DC) maturation, immune synapse formation, and subsequent T cell ...responses.
Methods
Here, we investigated the role of R‐Ras in allergen‐induced immune response (type 2 immune response) in Rras deficient (R‐Ras KO) and wild type (WT) mice.
Results
Initially, we found that the number of conventional DC's in the lymph nodes (LNs) was reduced in R‐Ras KO mice. The expression of co‐stimulatory CD80 and CD86 molecules on these cells was also reduced on DC's from the R‐Ras KO mice. However, there was no difference in papain‐induced immune response between the R‐Ras WT and KO as measured by serum IgE levels after the immunization. Interestingly, neither the DC number nor co‐stimulatory molecule expression was different between WT and R‐Ras KO animals after the immunization.
Conclusions
Taken together, despite having reduced number of conventional DC's in the R‐Ras KO mice and low expression of CD80 on DC's, the R‐Ras KO mice are capable of mounting papain‐induced IgE responses comparable to that of the WT mice. To our knowledge, this is the first report addressing potential differences in in vivo allergen responses regulated by the R‐Ras GTPase.
Deficiency of small GTPase—R‐Ras—has been shown to reduce immune synapse formation between T‐cell and dendritic cell (DC). Impaired T‐cell/DC interaction could skew immune responses toward Th2 direction. We show here, that R‐Ras deficient mice are capable of mounting normal type 2 immune response in vivo, as judged by IgE production in response to papain allergen immunization.
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