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• W3134732416 abstract "•VIMP is temporally upregulated after TCR stimulation in human CD4 effector T cells•VIMP inhibits cytokine expression in human CD4 effector T cells•VIMP inhibits cytokine expression via the NFATC2/Ca2+ signaling pathway•VIMP inhibits cytokine expression by controlling E2F5 expression Many players regulating the CD4+ T cell-mediated inflammatory response have already been identified. However, the critical nodes that constitute the regulatory and signaling networks underlying CD4 T cell responses are still missing. Using a correlation-network-guided approach, here we identified VIMP (VCP-interacting membrane protein), one of the 25 genes encoding selenoproteins in humans, as a gene regulating the effector functions of human CD4 T cells, especially production of several cytokines including IL2 and CSF2. We identified VIMP as an endogenous inhibitor of cytokine production in CD4 effector T cells via both the E2F5 transcription regulatory pathway and the Ca2+/NFATC2 signaling pathway. Our work not only indicates that VIMP might be a promising therapeutic target for various inflammation-associated diseases but also shows that our network-guided approach can significantly aid in predicting new functions of the genes of interest. Many players regulating the CD4+ T cell-mediated inflammatory response have already been identified. However, the critical nodes that constitute the regulatory and signaling networks underlying CD4 T cell responses are still missing. Using a correlation-network-guided approach, here we identified VIMP (VCP-interacting membrane protein), one of the 25 genes encoding selenoproteins in humans, as a gene regulating the effector functions of human CD4 T cells, especially production of several cytokines including IL2 and CSF2. We identified VIMP as an endogenous inhibitor of cytokine production in CD4 effector T cells via both the E2F5 transcription regulatory pathway and the Ca2+/NFATC2 signaling pathway. Our work not only indicates that VIMP might be a promising therapeutic target for various inflammation-associated diseases but also shows that our network-guided approach can significantly aid in predicting new functions of the genes of interest. CD4+ T cells represent a major subset of immune cells that are crucial for mounting and regulating an adequate immune response. However, during many infectious and complex chronic diseases, those T cells are dysregulated, either having an impaired responsive capacity or causing adverse effects through self-recognition and/or overactivation. Therefore, rebalancing the CD4+ T cell-mediated inflammatory response has been essential for the design of therapeutic options for those diseases (Zhu and Paul, 2010aZhu J. Paul W.E. Heterogeneity and plasticity of T helper cells.Cell Res. 2010; 20: 4-12Crossref PubMed Scopus (391) Google Scholar). Although many players regulating the inflammatory response, cytokine production, and differentiation of CD4+ T cells have already been identified in the past (Brownlie and Zamoyska, 2013Brownlie R.J. Zamoyska R. T cell receptor signalling networks: branched, diversified and bounded.Nat. Rev. Immunol. 2013; 13: 257-269Crossref PubMed Scopus (294) Google Scholar; Rodriguez-Jorge et al., 2019Rodriguez-Jorge O. Kempis-Calanis L.A. Abou-Jaoude W. Gutierrez-Reyna D.Y. Hernandez C. Ramirez-Pliego O. Thomas-Chollier M. Spicuglia S. Santana M.A. Thieffry D. Cooperation between T cell receptor and Toll-like receptor 5 signaling for CD4(+) T cell activation.Sci. Signal. 2019; 12: eaar3641Crossref PubMed Scopus (21) Google Scholar; Saez-Rodriguez et al., 2007Saez-Rodriguez J. Simeoni L. Lindquist J.A. Hemenway R. Bommhardt U. Arndt B. Haus U.U. Weismantel R. Gilles E.D. Klamt S. et al.A logical model provides insights into T cell receptor signaling.PLoS Comput. Biol. 2007; 3: e163Crossref PubMed Scopus (229) Google Scholar; Zhu and Paul, 2010bZhu J. Paul W.E. Peripheral CD4+ T-cell differentiation regulated by networks of cytokines and transcription factors.Immunol. Rev. 2010; 238: 247-262Crossref PubMed Scopus (373) Google Scholar), a thorough understanding of the regulatory and signaling networks governing inflammatory cytokine production in T cells is still missing. The gap is not only attributable to the long-standing nature of traditional trial-and-error experimental procedures but also to the lack of reliable high-throughput computational prediction. VIMP, also known as NCBI: Selenoprotein S (SELS), SELENOS, TANIS, or SEPS1, is one of the only 25 genes encoding the 21st amino acid selenocysteine in humans (Schomburg, 2011Schomburg L. Selenium, selenoproteins and the thyroid gland: interactions in health and disease.Nat. Rev. Endocrinol. 2011; 8: 160-171Crossref PubMed Scopus (191) Google Scholar). Located in the endoplasmic reticulum (ER) membrane, VIMP is mainly known as an important component of the ER-associated degradation (ERAD) complex (Kim et al., 2007Kim K.H. Gao Y. Walder K. Collier G.R. Skelton J. Kissebah A.H. SEPS1 protects RAW264.7 cells from pharmacological ER stress agent-induced apoptosis.Biochem. Biophys. Res. Commun. 2007; 354: 127-132Crossref PubMed Scopus (69) Google Scholar; Qin et al., 2016Qin H.S. Yu P.P. Sun Y. Wang D.F. Deng X.F. Bao Y.L. Song J. Sun L.G. Song Z.B. Li Y.X. Paclitaxel inhibits selenoprotein S expression and attenuates endoplasmic reticulum stress.Mol. Med. Rep. 2016; 13: 5118-5124Crossref PubMed Scopus (10) Google Scholar) and physically binds to several ER membrane proteins (Lee et al., 2015Lee J.H. Park K.J. Jang J.K. Jeon Y.H. Ko K.Y. Kwon J.H. Lee S.R. Kim I.Y. Selenoprotein S-dependent selenoprotein K binding to p97(VCP) protein is essential for endoplasmic reticulum-associated degradation.J. Biol. Chem. 2015; 290: 29941-29952Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar; Ye et al., 2005Ye Y. Shibata Y. Kikkert M. van Voorden S. Wiertz E. Rapoport T.A. Recruitment of the p97 ATPase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane.Proc. Natl. Acad. Sci. U S A. 2005; 102: 14132-14138Crossref PubMed Scopus (271) Google Scholar). VIMP plays a role in mediating retro-translocation of misfolded proteins from the ER lumen to the cytosol, where the ubiquitin-dependent proteasomal degradation takes place (Ye et al., 2004Ye Y. Shibata Y. Yun C. Ron D. Rapoport T.A. A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol.Nature. 2004; 429: 841-847Crossref PubMed Scopus (782) Google Scholar). Genome-wide association studies have shown that polymorphisms in the promoter region of VIMP are linked to a wide spectrum of common complex diseases, including cardiovascular disease (Alanne et al., 2007Alanne M. Kristiansson K. Auro K. Silander K. Kuulasmaa K. Peltonen L. Salomaa V. Perola M. Variation in the selenoprotein S gene locus is associated with coronary heart disease and ischemic stroke in two independent Finnish cohorts.Hum. Genet. 2007; 122: 355-365Crossref PubMed Scopus (90) Google Scholar), diabetes (Karlsson et al., 2004Karlsson H.K. Lake S. Koistinen H.A. Krook A. Relationship between serum amyloid A level and Tanis/SelS mRNA expression in skeletal muscle and adipose tissue from healthy and type 2 diabetic subjects.Diabetes. 2004; 53: 1424-1428Crossref PubMed Scopus (55) Google Scholar; Olsson et al., 2011Olsson M. Olsson B. Jacobson P. Thelle D.S. Bjorkegren J. Walley A. Froguel P. Carlsson L.M. Sjoholm K. Expression of the selenoprotein S (SELS) gene in subcutaneous adipose tissue and SELS genotype are associated with metabolic risk factors.Metabolism. 2011; 60: 114-120Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), cancer (Meplan et al., 2010Meplan C. Hughes D.J. Pardini B. Naccarati A. Soucek P. Vodickova L. Hlavata I. Vrana D. Vodicka P. Hesketh J.E. Genetic variants in selenoprotein genes increase risk of colorectal cancer.Carcinogenesis. 2010; 31: 1074-1079Crossref PubMed Scopus (117) Google Scholar; Shibata et al., 2009Shibata T. Arisawa T. Tahara T. Ohkubo M. Yoshioka D. Maruyama N. Fujita H. Kamiya Y. Nakamura M. Nagasaka M. et al.Selenoprotein S (SEPS1) gene -105G>A promoter polymorphism influences the susceptibility to gastric cancer in the Japanese population.BMC Gastroenterol. 2009; 9: 2Crossref PubMed Scopus (57) Google Scholar; Sutherland et al., 2010Sutherland A. Kim D.H. Relton C. Ahn Y.O. Hesketh J. Polymorphisms in the selenoprotein S and 15-kDa selenoprotein genes are associated with altered susceptibility to colorectal cancer.Genes Nutr. 2010; 5: 215-223Crossref PubMed Scopus (88) Google Scholar), sepsis (He et al., 2014He L. Wang B. Yao Y. Su M. Ma H. Jia N. Protective effects of the SEPS1 gene on lipopolysaccharide-induced sepsis.Mol. Med. Rep. 2014; 9: 1869-1876Crossref PubMed Scopus (16) Google Scholar), and autoimmune diseases (Santos et al., 2014Santos L.R. Duraes C. Mendes A. Prazeres H. Alvelos M.I. Moreira C.S. Canedo P. Esteves C. Neves C. Carvalho D. et al.A polymorphism in the promoter region of the selenoprotein S gene (SEPS1) contributes to Hashimoto's thyroiditis susceptibility.J. Clin. Endocrinol. Metab. 2014; 99: E719-E723Crossref PubMed Scopus (44) Google Scholar; Seiderer et al., 2007Seiderer J. Dambacher J. Kuhnlein B. Pfennig S. Konrad A. Torok H.P. Haller D. Goke B. Ochsenkuhn T. Lohse P. et al.The role of the selenoprotein S (SELS) gene -105G>A promoter polymorphism in inflammatory bowel disease and regulation of SELS gene expression in intestinal inflammation.Tissue Antigens. 2007; 70: 238-246Crossref PubMed Scopus (34) Google Scholar), in which activation of the immune system is believed to be dysregulated (Kuchroo et al., 2012Kuchroo V.K. Ohashi P.S. Sartor R.B. Vinuesa C.G. Dysregulation of immune homeostasis in autoimmune diseases.Nat. Med. 2012; 18: 42-47Crossref PubMed Scopus (75) Google Scholar). Meanwhile, dysfunction of the ER and the unfolded protein response causes intestinal inflammatory diseases in several murine models (McGuckin et al., 2010McGuckin M.A. Eri R.D. Das I. Lourie R. Florin T.H. ER stress and the unfolded protein response in intestinal inflammation.Am. J. Physiol. Gastrointest. Liver Physiol. 2010; 298: G820-G832Crossref PubMed Scopus (129) Google Scholar). Additionally, a reduced expression of VIMP causes an increased expression of inflammatory cytokines, such as NCBI: IL6, IL1β, and TNFα in macrophages (Curran et al., 2005Curran J.E. Jowett J.B. Elliott K.S. Gao Y. Gluschenko K. Wang J. Abel Azim D.M. Cai G. Mahaney M.C. Comuzzie A.G. et al.Genetic variation in selenoprotein S influences inflammatory response.Nat. Genet. 2005; 37: 1234-1241Crossref PubMed Scopus (298) Google Scholar), as well as IL1β and IL6 expression in astrocytes (Fradejas et al., 2011Fradejas N. Serrano-Perez Mdel C. Tranque P. Calvo S. Selenoprotein S expression in reactive astrocytes following brain injury.Glia. 2011; 59: 959-972Crossref PubMed Scopus (42) Google Scholar). However, other studies did not show significant association between VIMP and the examined human inflammatory diseases (Martinez et al., 2008Martinez A. Santiago J.L. Varade J. Marquez A. Lamas J.R. Mendoza J.L. de la Calle H. Diaz-Rubio M. de la Concha E.G. Fernandez-Gutierrez B. et al.Polymorphisms in the selenoprotein S gene: lack of association with autoimmune inflammatory diseases.BMC Genomics. 2008; 9: 329Crossref PubMed Scopus (33) Google Scholar). This controversy underlines the necessity for a better understanding of how VIMP contributes to the pathogenesis of some inflammatory diseases, i.e., through which cell types and which molecular pathways VIMP contributes to the observed dysregulated inflammatory responses. Therefore, we sought to investigate whether and how VIMP plays a role in relevant specific immune cells, e.g., CD4+ T cells, a key subset of immune cells orchestrating different types of immune responses and being heavily involved in different complex diseases, as well as infectious diseases, such as COVID-19 (Braun et al., 2020Braun J. Loyal L. Frentsch M. Wendisch D. Georg P. Kurth F. Hippenstiel S. Dingeldey M. Kruse B. Fauchere F. et al.SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19.Nature. 2020; 587: 270-274Crossref PubMed Scopus (483) Google Scholar; Mathew et al., 2020Mathew D. Giles J.R. Baxter A.E. Oldridge D.A. Greenplate A.R. Wu J.E. Alanio C. Kuri-Cervantes L. Pampena M.B. D’Andrea K. et al.Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications.Science. 2020; 369: eabc8511Crossref PubMed Google Scholar). We have previously developed a correlation-network-guided approach, based on the guilt-by-association theory (Beyer et al., 2007Beyer A. Bandyopadhyay S. Ideker T. Integrating physical and genetic maps: from genomes to interaction networks.Nat. Rev. Genet. 2007; 8: 699-710Crossref PubMed Scopus (159) Google Scholar; Gillis and Pavlidis, 2011Gillis J. Pavlidis P. The role of indirect connections in gene networks in predicting function.Bioinformatics. 2011; 27: 1860-1866Crossref PubMed Scopus (44) Google Scholar; Oliver, 2000Oliver S. Guilt-by-association goes global.Nature. 2000; 403: 601-603Crossref PubMed Scopus (357) Google Scholar), to predict novel key genes of a given biological process or function and have successfully applied it to human CD4+CD25highCD127low regulatory T cells (Tregs) (Danileviciute et al., 2019Danileviciute E. Zeng N. Capelle C. Paczia N. Gillespie M.A. Kurniawan H. Coowar D. Vogt Weisenhorn D.M. Giro G.G. Grusdat M. et al.PARK7/DJ-1 promotes pyruvate dehydrogenase activity and maintains Treg homeostasis.https://doi.org/10.1101/2019.12.20.884809Date: 2019Google Scholar; He et al., 2012He F. Chen H. Probst-Kepper M. Geffers R. Eifes S. Del Sol A. Schughart K. Zeng A.P. Balling R. PLAU inferred from a correlation network is critical for suppressor function of regulatory T cells.Mol. Syst. Biol. 2012; 8: 624Crossref PubMed Scopus (36) Google Scholar). Here, we extended the strategy to human CD4+ effector T cells (Teffs) that were derived and expanded from sorted CD4+CD25- T cells by co-culturing with EBV-transformed B cells and were able to predict that VIMP might play an important role in regulating the effector responses of Teffs. Combining both the network analysis and experimental verification, we identify VIMP as a previously unreported vital endogenous inhibitor of cytokine production in human CD4+ Teffs and reveal the molecular mechanisms through which VIMP regulates CD4+ Teff responses. Using our previously reported high-time-resolution (HTR) time-series transcriptome data of Tregs and Teffs following TCR (T cell receptor) stimulation in the first 6 h (He et al., 2012He F. Chen H. Probst-Kepper M. Geffers R. Eifes S. Del Sol A. Schughart K. Zeng A.P. Balling R. PLAU inferred from a correlation network is critical for suppressor function of regulatory T cells.Mol. Syst. Biol. 2012; 8: 624Crossref PubMed Scopus (36) Google Scholar), we observed that the transcript level of VIMP in Teffs temporally peaked within 2–3 h following stimulation, which was followed by a gradual decrease (Figure 1A). In contrast, the VIMP mRNA level was kept almost constant in Tregs during the first 6 h following TCR stimulation (Figure 1A), indicating a possible specific role for VIMP in Teffs. Our quantitative real-time PCR (qPCR) results validated the transitionally elevated expression of the VIMP transcript in Teffs isolated from different healthy donors (Figure 1B). We also observed a correlation over time between the transcription levels of NCBI: VIMP, IL2, IL13, and CSF2 (GM-CSF) following TCR stimulation, indicating a potential regulatory relationship between VIMP and some of the cytokines in Teffs (Figure 1B). By flow cytometry (Figure 1C), we confirmed the gradual upregulation of VIMP protein expression in the first 5 h following TCR stimulation. In summary, both mRNA and protein expression of VIMP were upregulated following TCR stimulation, which was correlated with the expression of several examined cytokines, indicating a potential role of VIMP in regulating Teff responses. The upregulation of VIMP and its correlation to cytokine expression encouraged us to further investigate VIMP's potential role in CD4 T cell responses. We and others have previously shown that the enriched pathways, processes or functions among the genes surrounding a given hub gene in the correlation network might give valuable indications on potential new functions of the given hub gene (Danileviciute et al., 2019Danileviciute E. Zeng N. Capelle C. Paczia N. Gillespie M.A. Kurniawan H. Coowar D. Vogt Weisenhorn D.M. Giro G.G. Grusdat M. et al.PARK7/DJ-1 promotes pyruvate dehydrogenase activity and maintains Treg homeostasis.https://doi.org/10.1101/2019.12.20.884809Date: 2019Google Scholar; He et al., 2012He F. Chen H. Probst-Kepper M. Geffers R. Eifes S. Del Sol A. Schughart K. Zeng A.P. Balling R. PLAU inferred from a correlation network is critical for suppressor function of regulatory T cells.Mol. Syst. Biol. 2012; 8: 624Crossref PubMed Scopus (36) Google Scholar). Therefore, we used our correlation network-guided approach to predict the potential functions of VIMP by identifying the enriched pathways among the genes that are linked to VIMP within the subnetwork of the Teff correlation network, which was extracted from our published HTR datasets and networks (He et al., 2012He F. Chen H. Probst-Kepper M. Geffers R. Eifes S. Del Sol A. Schughart K. Zeng A.P. Balling R. PLAU inferred from a correlation network is critical for suppressor function of regulatory T cells.Mol. Syst. Biol. 2012; 8: 624Crossref PubMed Scopus (36) Google Scholar) (Figure 2A). Consistent with its known function and its localization in the ER membrane, the genes surrounding VIMP in the correlation network were significantly enriched for ER components (p value = 1.7 × 10−7, cumulative binomial distribution) (Figure 2A). Furthermore, 3 of the 10 experimentally validated VIMP-binding partners found in the literature in other cellular types are directly linked to VIMP in the Teff correlation network (p value = 2.0 × 10−4, http://string-db.org [Szklarczyk et al., 2019Szklarczyk D. Gable A.L. Lyon D. Junge A. Wyder S. Huerta-Cepas J. Simonovic M. Doncheva N.T. Morris J.H. Bork P. et al.STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets.Nucleic Acids Res. 2019; 47: D607-D613Crossref PubMed Scopus (4863) Google Scholar]), indicating the reliability of our method. Surprisingly, the pathway enrichment analysis shows that the genes linked to VIMP are significantly enriched for components involved in the TCR signaling pathway (p value = 1.2 × 10−3, cumulative binomial distribution) (Figure 2A), suggesting a potential role of VIMP in the Teff response according to our network-based analysis strategy (Danileviciute et al., 2019Danileviciute E. Zeng N. Capelle C. Paczia N. Gillespie M.A. Kurniawan H. Coowar D. Vogt Weisenhorn D.M. Giro G.G. Grusdat M. et al.PARK7/DJ-1 promotes pyruvate dehydrogenase activity and maintains Treg homeostasis.https://doi.org/10.1101/2019.12.20.884809Date: 2019Google Scholar; He et al., 2012He F. Chen H. Probst-Kepper M. Geffers R. Eifes S. Del Sol A. Schughart K. Zeng A.P. Balling R. PLAU inferred from a correlation network is critical for suppressor function of regulatory T cells.Mol. Syst. Biol. 2012; 8: 624Crossref PubMed Scopus (36) Google Scholar). However, the genes linked to the hub gene of interests in the correlation network could follow at least two scenarios (He and Ollert, 2016He F.Q. Ollert M. Network-guided key gene discovery for a given cellular process.Adv. Biochem. Eng. Biotechnol. 2016; PubMed Google Scholar; Langfelder and Horvath, 2008Langfelder P. Horvath S. WGCNA: an R package for weighted correlation network analysis.BMC Bioinformatics. 2008; 9: 559Crossref PubMed Scopus (7625) Google Scholar; van Dam et al., 2018van Dam S. Vosa U. van der Graaf A. Franke L. de Magalhaes J.P. Gene co-expression analysis for functional classification and gene-disease predictions.Brief Bioinform. 2018; 19: 575-592PubMed Google Scholar). First, those genes could be co-regulated by chance with the hub gene and perform independent functions. Second, those genes could be co-expressed with the hub gene and play related roles in the same pathways to coordinate cellular resources for a particular function or purpose under certain conditions. We will test these possibilities in this work. To systematically assess whether and how VIMP controls the inflammatory response of Teffs, we performed a transcriptome analysis of CD4 Teffs isolated from peripheral blood mononuclear cells (PBMCs) of three healthy donors that were subjected to a specific-small interfering RNA (siRNA) knockdown of VIMP (si_VIMP) or a control unspecific scrambled siRNA (si_NS) followed by anti-CD3/-CD28 stimulation (Figure 2B). As shown in Figure 2C, the mRNA expression of VIMP was significantly downregulated in Teffs by using siRNA knockdown. As VIMP has reported functions in ER stress, we first checked the ER-stress responsive genes in the transcriptomic data of the Teffs transfected with si_VIMP versus that treated with control siRNA (si_NS). By perturbing the expression of VIMP, we expected a change in the expression of some ER-stress responsive genes. Nonetheless, our transcriptome data of Teffs with VIMP partial knockdown did not show any significant change in mRNA expression of those genes (e.g., NCBI: CHOP [DDIT3], GRP78 [HSPA5], EDEM1, DNAJC3 [P58IPK], and DNAJB9 [ERdj4] [Lee et al., 2003Lee A.H. Iwakoshi N.N. Glimcher L.H. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response.Mol. Cell Biol. 2003; 23: 7448-7459Crossref PubMed Scopus (1527) Google Scholar]) (Figure 2D). Only the expression of the ER-stress regulator XBP1 (Yoshida et al., 2001Yoshida H. Matsui T. Yamamoto A. Okada T. Mori K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor.Cell. 2001; 107: 881-891Abstract Full Text Full Text PDF PubMed Scopus (2784) Google Scholar) was significantly but modestly decreased. Indeed, data from intestinal epithelial cells show that VIMP is only a marker, but not a regulator, of ER stress (Speckmann et al., 2014Speckmann B. Gerloff K. Simms L. Oancea I. Shi W. McGuckin M.A. Radford-Smith G. Khanna K.K. Selenoprotein S is a marker but not a regulator of endoplasmic reticulum stress in intestinal epithelial cells.Free Radic. Biol. Med. 2014; 67: 265-277Crossref PubMed Scopus (28) Google Scholar). This shows that the direct involvement of VIMP in ER stress might not be ubiquitous to all cell types. We therefore ruled out the possibility that VIMP directly regulates the expression of the ER-stress responsive genes, indicating other roles of VIMP in modulating the Teff responses. Considering that the TCR signaling pathways were significantly enriched in the VIMP correlation network, we further analyzed the genes related to the TCR signaling pathway in Teffs after VIMP knockdown. Notably, we found 13 significantly upregulated genes involved in the TCR signaling, including several cytokines, namely, NCBI: IL2, IL4, CSF2, and IFNG (refer to https://www.genome.jp/kegg-bin/show_pathway?hsa04660) in the microarray datasets of the Teffs, although only subjected to a partial knockdown of VIMP (Figure 2E). Moreover, transcripts of the key TCR-related signaling molecules, such as NCBI: GRAP2, ZAP70, RASGRP1, and RAF1, were significantly affected (Figure 2E). With the observation in mind that VIMP and the TCR signaling-related genes were directly linked in our HTR correlation network (Figure 2A), this effect of the siRNA perturbation was not unexpected. Our results suggest that VIMP negatively regulates the expression of specific cytokines and influences the expression of important components of the TCR signaling pathway. To further confirm whether VIMP regulates cytokine expression in Teffs, using PBMC of independent donors we measured the cytokine mRNA expression by qPCR and the secreted cytokines of Teffs that were exposed to a VIMP knockdown. Indeed, NCBI: IL2, IL21, and CSF2 mRNA were significantly upregulated in stimulated Teffs transfected with si_VIMP, compared with control Teffs (with si_NS) (Figure 2F). This observation was further consolidated by increased IL2, IL21, and GM-CSF protein levels in the culture media of stimulated Teffs transfected with si_VIMP, compared with that treated with control scrambled siRNA (Figure 2G). Furthermore, the VIMP knockdown also significantly promoted T cell proliferation as indicated by both carboxyfluorescein succinimidyl ester (CFSE) peak shifting and Teff cell number counting experiments (Figure 2H and 2I). As IL2 concentration was already significantly higher at 3 h following stimulation (Figure 2G) and no cell division was expected, the enhanced IL2 secretion following VIMP knockdown was not simply caused by more Teffs. All the analyses were done under the precondition that VIMP protein was successfully silenced (Figure 2J). In short, VIMP negatively regulates the expression of several cytokines in Teffs following stimulation. Considering that VIMP encodes selenocysteine, thus requiring selenium (Se) for its protein synthesis, and the fact that a relatively low concentration of Se was used in our T cell media (IMDM, ∼0.066 μM), we next supplemented sodium selenite in the T-cell culture media to the range of physiological concentrations (∼1 μM) (Rauhamaa et al., 2008Rauhamaa P. Kantola M. Viitak A. Kaasik T. Mussalo-Rauhamaa H. Selenium levels of Estonians.Eur. J. Clin. Nutr. 2008; 62: 1075-1078Crossref PubMed Scopus (8) Google Scholar; Safaralizadeh et al., 2005Safaralizadeh R. Kardar G.A. Pourpak Z. Moin M. Zare A. Teimourian S. Serum concentration of selenium in healthy individuals living in Tehran.Nutr. J. 2005; 4: 32Crossref PubMed Scopus (72) Google Scholar; Stranges et al., 2011Stranges S. Tabák A.G. Guallar E. Rayman M.P. Akbaraly T.N. Laclaustra M. Alfthan G. Mussalo-Rauhamaa H. Viikari J.S. Raitakari O.T. et al.Selenium status and blood lipids: the cardiovascular risk in Young Finns study.J. Intern. Med. 2011; 270: 469-477Crossref PubMed Scopus (27) Google Scholar). In line with the reported inverse association between Se status and inflammatory bowel diseases (Kudva et al., 2015Kudva A.K. Shay A.E. Prabhu K.S. Selenium and inflammatory bowel disease.Am. J. Physiol. Gastrointest. Liver Physiol. 2015; 309: G71-G77Crossref PubMed Scopus (72) Google Scholar), increasing the concentration of Se in the media generated a dose-dependent suppressive effect on IL2 production of sorted CD4 Teffs following TCR stimulation (Figures S1A and S1B). Meanwhile, increasing Se concentration to a physiological concentration upregulated the VIMP expression of stimulated CD4 Teffs in three of five tested donors (Figures S1C and S1D). These results again indicate that Se, at least partially, negatively regulates the expression of cytokines, e.g., IL2 in CD4 Teffs via VIMP, which is in line with our knockdown results. Next, we aimed to identify any (co-)transcription factors (TFs), whose expression were significantly affected after silencing VIMP, as they often serve as the key components orchestrating the activity of the relevant pathways. Through a systematic analysis of all the known mammalian TFs or co-factors (Zhang et al., 2012Zhang H.M. Chen H. Liu W. Liu H. Gong J. Wang H. Guo A.Y. AnimalTFDB: a comprehensive animal transcription factor database.Nucleic Acids Res. 2012; 40: D144-D149Crossref PubMed Scopus (209) Google Scholar) in our microarray datasets, NCBI: E2F5 was found to be the most significantly upregulated TF, following a partial VIMP knockdown (Figure 3A). Conversely, NCBI: RNF14 (ring finger protein 14) was the most downregulated cofactor together with the downregulated TFs NCBI: CEBPG (CCAAT enhancer binding protein gamma), NCBI: ZBTB20 (zinc finger and BTB domain containing 20), and NCBI: IRX3 (Iroquois homeobox 3) (Figure 3A). We further confirmed the expression change of these (co-)TFs by qPCR in independent healthy donors (Figure 3B). E2F5 has been reported to be a downstream target of IL-2 in an immortalized human T cell line (Brennan et al., 1997Brennan P. Babbage J.W. Burgering B.M. Groner B. Reif K. Cantrell D.A. Phosphatidylinositol 3-kinase couples the interleukin-2 receptor to the cell cycle regulator E2F.Immunity. 1997; 7: 679-689Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). But to our knowledge, there are no reports yet of E2F5 sitting at the upstream pathways regulating inflammatory responses, especially cytokine production. Nevertheless, being the most significantly upregulated TF following a partial knockdown of VIMP, we assumed that E2F5 might be an important component in the regulatory pathway through which VIMP regulates the Teff inflammatory response. Therefore, we decided to investigate whether VIMP controls the cytokine expression by negatively regulating E2F5 expression in stimulated Teffs. To examine this hypothesis, we silenced VIMP alone or in combination with E2F5 and measured the expression of selected cytokines by qPCR. In addition to the reduced expression of VIMP, the upregulation of E2F5 expression that was driven by VIMP knockdown was abolished in the VIMP and E2F5 double knockdown Teffs (Figure 3C). Silencing VIMP alone upregulated IL2 expression in stimulated Teffs, whereas a dual knockdown of VIMP and E2F5 suppressed the surge of IL2 caused by VIMP knockdown alone (Figure 3C). Even though E2F5 is a general regulator of transcription, we did not observe any effect of E2F5 knockdown on genes that are not directly involved in Teff inflammatory response, such as NCBI: CTLA4 (Figure 3C). This ex" @default.
• W3134732416 created "2021-03-15" @default.
• W3134732416 creator A5008785148 @default.
• W3134732416 creator A5024581567 @default.
• W3134732416 creator A5033427289 @default.
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• W3134732416 date "2021-04-01" @default.
• W3134732416 modified "2023-10-14" @default.
• W3134732416 title "Identification of VIMP as a gene inhibiting cytokine production in human CD4+ effector T cells" @default.
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