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Lupus-Associated Functional Polymorphism in PNP Causes Cell Cycle Abnormalities and Interferon Pathway Activation in Human Immune Cells.

ABSTRACT: Systemic lupus erythematosus (SLE) is frequently characterized by activation of the type I interferon (IFN) pathway. We previously observed that a missense single-nucleotide polymorphism (rs1049564) in the purine nucleoside phosphorylase (PNP) gene was associated with high levels of IFN in SLE. PNP is a key enzyme involved in purine metabolism. In this study, we performed functional follow-up of this polymorphism in human cells.Type I IFN was measured in patient sera, using a reporter cell assay. Structural modeling of the PNP variant was performed using PyMOL software. PNP messenger RNA (mRNA) and protein levels and type I IFN-induced gene expression were measured in lymphoblastoid cell lines with known PNP rs1049564 genotypes. The cell cycle was assayed using flow cytometry.Structural modeling indicated no major disruption in folding related to rs1049564. We observed that homozygous rs1049564 TT lymphoblastoid cells had decreased PNP mRNA expression and protein levels, and that cells with the TT genotype had reduced PNP enzymatic activity even when the amount of PNP was controlled. Cells with the TT genotype had a 2-fold increase in S-phase block as compared with cells with the homozygous CC phenotype. The S-phase block could be pharmacologically reversed with hypoxanthine and adenosine, supporting the notion that relative PNP deficiency is the cause of the S-phase block. Type I IFN-induced transcripts were increased in a dose-response manner related to the rs1049564 T allele, at both baseline and after type I IFN stimulation.The PNP rs1049564 T allele is a loss-of-function variant that induces S-phase block and IFN pathway activation in lymphocytes. The S-phase block could be rescued in our in vitro experiments, suggesting the potential for personalized treatment.

SUBMITTER: Ghodke-Puranik Y

PROVIDER: S-EPMC5711527 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Lupus-Associated Functional Polymorphism in PNP Causes Cell Cycle Abnormalities and Interferon Pathway Activation in Human Immune Cells.

Ghodke-Puranik Yogita Y Dorschner Jessica M JM Vsetecka Danielle M DM Amin Shreyasee S Makol Ashima A Ernste Floranne F Osborn Thomas T Moder Kevin K Chowdhary Vaidehi V Eliopoulos Elias E Zervou Maria I MI Goulielmos George N GN Jensen Mark A MA Niewold Timothy B TB

Arthritis & rheumatology (Hoboken, N.J.) 20171109 12

<h4>Objective</h4>Systemic lupus erythematosus (SLE) is frequently characterized by activation of the type I interferon (IFN) pathway. We previously observed that a missense single-nucleotide polymorphism (rs1049564) in the purine nucleoside phosphorylase (PNP) gene was associated with high levels of IFN in SLE. PNP is a key enzyme involved in purine metabolism. In this study, we performed functional follow-up of this polymorphism in human cells.<h4>Methods</h4>Type I IFN was measured in patient ...[more]

PMID: 28859258

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Project description:Type I interferons (IFNs), mostly IFNα and IFNβ, and the type I IFN Signature are important in the pathogenesis of Systemic Lupus Erythematosus (SLE), an autoimmune chronic condition linked to inflammation. Both IFNα and IFNβ trigger a signaling cascade that, through the activation of JAK1, TYK2, STAT1 and STAT2, initiates gene transcription of IFN stimulated genes (ISGs). Noteworthy, other STAT family members and IFN Responsive Factors (IRFs) can also contribute to the activation of the IFN response. Aberrant type I IFN signaling, therefore, can exacerbate SLE by deregulated homeostasis leading to unnecessary persistence of the biological effects of type I IFNs. The etiopathogenesis of SLE is partially known and considered multifactorial. Family-based and genome wide association studies (GWAS) have identified genetic and transcriptional abnormalities in key molecules directly involved in the type I IFN signaling pathway, namely TYK2, STAT1 and STAT4, and IRF5. Gain-of-function mutations that heighten IFNα/β production, which in turn maintains type I IFN signaling, are found in other pathologies like the interferonopathies. However, the distinctive characteristics have yet to be determined. Signaling molecules activated in response to type I IFNs are upregulated in immune cell subsets and affected tissues of SLE patients. Moreover, Type I IFNs induce chromatin remodeling leading to a state permissive to transcription, and SLE patients have increased global and gene-specific epigenetic modifications, such as hypomethylation of DNA and histone acetylation. Epigenome wide association studies (EWAS) highlight important differences between SLE patients and healthy controls in Interferon Stimulated Genes (ISGs). The combination of environmental and genetic factors may stimulate type I IFN signaling transiently and produce long-lasting detrimental effects through epigenetic alterations. Substantial evidence for the pathogenic role of type I IFNs in SLE advocates the clinical use of neutralizing anti-type I IFN receptor antibodies as a therapeutic strategy, with clinical studies already showing promising results. Current and future clinical trials will determine whether drugs targeting molecules of the type I IFN signaling pathway, like non-selective JAK inhibitors or specific TYK2 inhibitors, may benefit people living with lupus.

Project description:Enhancer of zeste homolog 2 (EZH2) is a histone-lysine N-methyltransferase mediating trimethylation of H3K27, which represses gene expression and is critical to immune regulation. Inhibition of EZH2 is proved to have the potential of treating many diseases. However, whether inhibition of EZH2 affects type I interferon (IFN-I) signaling pathway, the abnormality of which is an important pathogenic mechanism for SLE, is still elusive. Here, we report, unexpectedly, a positive regulatory function of EZH2 in IFN-I signaling pathway, which contributes to the overactivation of IFN-I signaling pathway in SLE. We show that the expression of EZH2 was upregulated and positively correlated with the overexpression of interferon stimulated genes (ISGs) in both peripheral blood mononuclear cells and renal tissues of SLE patients. In vitro inhibition of EZH2 by either siRNAs or chemical inhibitors reduced the phosphorylation of STAT1 and the induction of ISGs stimulated by IFN-I. Additionally, inhibition of EZH2 interfered with the in vivo and ex vivo activation of IFN-I signaling pathway elicited by intravenous injection of adenovirus vector expressing mouse IFN-α5 and exogeneous stimulation with IFN-α, respectively. We evaluated the therapeutic effects of EZH2 inhibitor in NZB/NZW F1 mice which depend on IFN-I signaling pathway for the lupus-like disease development. Administration of EZH2 inhibitor prolonged the survival, reduced the levels of anti-dsDNA autoantibodies, and improved lupus nephritis of the mice. What's more, EZH2 inhibitor attenuated the expression of ISGs in the kidneys of these mice. In summary, we show that excessive EZH2 contributes to the overactivation of IFN-I signaling pathway in SLE. EZH2 inhibitor has the potential to inhibit IFN-I signaling pathway and alleviate lupus nephritis. Additionally, diverse disease driving pathways exist among systemic lupus erythematosus (SLE) patient, and even in the same patients. Common regulators of different pathogenic pathways can be multivalent therapeutic targets. Together with previous studies showing EZH2 is involved in T-cell and B-cell mediated immune responses, EZH2 could be a potent multivalent therapeutic target for SLE.

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Lupus-Associated Functional Polymorphism in PNP Causes Cell Cycle Abnormalities and Interferon Pathway Activation in Human Immune Cells.

Publications

Lupus-Associated Functional Polymorphism in PNP Causes Cell Cycle Abnormalities and Interferon Pathway Activation in Human Immune Cells.

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