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• W2492545900 abstract "Inflammation and endoplasmic reticulum (ER) stress are associated with many neurological diseases. ER stress is brought on by the accumulation of misfolded proteins in the ER, which leads to activation of the unfolded protein response (UPR), a conserved pathway that transmits signals to restore homeostasis or eliminate the irreparably damaged cell. We provide evidence that inhibition or genetic haploinsufficiency of protein kinase R-like endoplasmic reticulum kinase (PERK) can selectively control inflammation brought on by ER stress without impinging on UPR-dependent survival and adaptive responses or normal immune responses. Using astrocytes lacking one or both alleles of PERK or the PERK inhibitor GSK2606414, we demonstrate that PERK haploinsufficiency or partial inhibition led to reduced ER stress-induced inflammation (IL-6, CCL2, and CCL20 expression) without compromising prosurvival responses. In contrast, complete loss of PERK blocked canonical PERK-dependent UPR genes and promoted apoptosis. Reversal of eIF2α-mediated translational repression using ISRIB potently suppressed PERK-dependent inflammatory gene expression, indicating that the selective modulation of inflammatory gene expression by PERK inhibition may be linked to attenuation of eIF2α phosphorylation and reveals a previously unknown link between translational repression and transcription of inflammatory genes. Additionally, ER-stressed astrocytes can drive an inflammatory M1-like phenotype in microglia, and this can be attenuated with inhibition of PERK. Importantly, targeting PERK neither disrupted normal cytokine signaling in astrocytes or microglia nor impaired macrophage phagocytosis or T cell polarization. Collectively, this work suggests that targeting PERK may provide a means for selective immunoregulation in the context of ER stress without disrupting normal immune function. Inflammation and endoplasmic reticulum (ER) stress are associated with many neurological diseases. ER stress is brought on by the accumulation of misfolded proteins in the ER, which leads to activation of the unfolded protein response (UPR), a conserved pathway that transmits signals to restore homeostasis or eliminate the irreparably damaged cell. We provide evidence that inhibition or genetic haploinsufficiency of protein kinase R-like endoplasmic reticulum kinase (PERK) can selectively control inflammation brought on by ER stress without impinging on UPR-dependent survival and adaptive responses or normal immune responses. Using astrocytes lacking one or both alleles of PERK or the PERK inhibitor GSK2606414, we demonstrate that PERK haploinsufficiency or partial inhibition led to reduced ER stress-induced inflammation (IL-6, CCL2, and CCL20 expression) without compromising prosurvival responses. In contrast, complete loss of PERK blocked canonical PERK-dependent UPR genes and promoted apoptosis. Reversal of eIF2α-mediated translational repression using ISRIB potently suppressed PERK-dependent inflammatory gene expression, indicating that the selective modulation of inflammatory gene expression by PERK inhibition may be linked to attenuation of eIF2α phosphorylation and reveals a previously unknown link between translational repression and transcription of inflammatory genes. Additionally, ER-stressed astrocytes can drive an inflammatory M1-like phenotype in microglia, and this can be attenuated with inhibition of PERK. Importantly, targeting PERK neither disrupted normal cytokine signaling in astrocytes or microglia nor impaired macrophage phagocytosis or T cell polarization. Collectively, this work suggests that targeting PERK may provide a means for selective immunoregulation in the context of ER stress without disrupting normal immune function. Normal inflammatory responses are transient, protective, and essential to overall health. However, chronic inflammation is associated with a multitude of common maladies, including obesity, diabetes, cancer, and neurodegeneration. Left unchecked, inflammation can cause cellular dysfunction and tissue damage from the prolonged production of reactive oxygen/nitrogen species and proteases, among others, potentially exacerbating the disease state (1Medzhitov R. Origin and physiological roles of inflammation.Nature. 2008; 454: 428-435Crossref PubMed Scopus (3887) Google Scholar). Broadly targeting inflammatory processes with drugs such as glucocorticoids can be difficult in chronic conditions because of serious side effects (2Coutinho A.E. Chapman K.E. The anti-inflammatory and immunosuppressive effects of glucocorticoids, recent developments and mechanistic insights.Mol. Cell Endocrinol. 2011; 335: 2-13Crossref PubMed Scopus (963) Google Scholar) and may impair normal inflammatory processes that are essential for tissue repair and resolution (3Buckley C.D. Gilroy D.W. Serhan C.N. Stockinger B. Tak P.P. The resolution of inflammation.Nat. Rev. Immunol. 2013; 13: 59-66Crossref PubMed Scopus (273) Google Scholar, 4Raposo C. Graubardt N. Cohen M. Eitan C. London A. Berkutzki T. Schwartz M. CNS repair requires both effector and regulatory T cells with distinct temporal and spatial profiles.J. Neurosci. 2014; 34: 10141-10155Crossref PubMed Scopus (99) Google Scholar). The delicate balance between beneficial and detrimental inflammation is of profound importance in the CNS, where damage can have dire and long-lasting effects. Within the CNS, glial cells, particularly astrocytes and microglia, have a key role in controlling the inflammatory environment (5Ransohoff R.M. Brown M.A. Innate immunity in the central nervous system.J. Clin. Invest. 2012; 122: 1164-1171Crossref PubMed Scopus (703) Google Scholar, 6Burda J.E. Sofroniew M.V. Reactive gliosis and the multicellular response to CNS damage and disease.Neuron. 2014; 81: 229-248Abstract Full Text Full Text PDF PubMed Scopus (873) Google Scholar). Glial cells are in an inflammatory (gliotic) state in most neurological diseases. As with peripheral inflammatory responses, gliosis, when properly executed and resolved, is protective and promotes homeostasis (7Verkhratsky A. Parpura V. Pekna M. Pekny M. Sofroniew M. Glia in the pathogenesis of neurodegenerative diseases.Biochem. Soc. Trans. 2014; 42: 1291-1301Crossref PubMed Scopus (117) Google Scholar). However, chronic glia-derived inflammation may contribute to neurodegeneration (8Heneka M.T. Kummer M.P. Latz E. Innate immune activation in neurodegenerative disease.Nat. Rev. Immunol. 2014; 14: 463-477Crossref PubMed Scopus (857) Google Scholar). Conversely, a reduction of immunological function and surveillance in the CNS can be equally disruptive and may worsen injuries, impair cognitive function, and contribute to neurological diseases (9Bush T.G. Puvanachandra N. Horner C.H. Polito A. Ostenfeld T. Svendsen C.N. Mucke L. Johnson M.H. Sofroniew M.V. Leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice.Neuron. 1999; 23: 297-308Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar, 10Schwartz M. Kipnis J. Rivest S. Prat A. How do immune cells support and shape the brain in health, disease, and aging?.J. Neurosci. 2013; 33: 17587-17596Crossref PubMed Scopus (204) Google Scholar). Thus, a major challenge is to identify processes driving chronic damaging inflammation that can be targeted without disruption of normal beneficial immune function. Cell and tissue damage can elicit an inflammatory response (11Chen G.Y. Nuñez G. Sterile inflammation: sensing and reacting to damage.Nat. Rev. Immunol. 2010; 10: 826-837Crossref PubMed Scopus (2045) Google Scholar). In neurodegenerative diseases, cells are likely damaged through several processes, including the accumulation of misfolded proteins, leading to endoplasmic reticulum (ER) 3The abbreviations used are: ERendoplasmic reticulumUPRunfolded protein responsePERKprotein kinase R-like endoplasmic reticulum kinaseATFactivating transcription factorOSMOncostatin MqPCRquantitative PCRthapsthapsigargintunictunicamycinCHOPC/EBP homologous proteinC/EBPCCAAT/enhancer-binding protein. stress (12Hetz C. Mollereau B. Disturbance of endoplasmic reticulum proteostasis in neurodegenerative diseases.Nat. Rev. Neurosci. 2014; 15: 233-249Crossref PubMed Scopus (487) Google Scholar). ER stress activates the unfolded protein response (UPR), a homeostatic response to the accumulation of misfolded proteins in the ER. The UPR is mediated by trans-ER membrane proteins that include PERK, inositol-requiring enzyme 1 (IRE1), and activating transcription factor (ATF) 6. This response promotes reduced protein synthesis through PERK-dependent phosphorylation of eukaryotic initiation factor 2α (eIF2α) and selective up-regulation of transcriptional regulators and molecular chaperones to restore homeostasis (13Ron D. Walter P. Signal integration in the endoplasmic reticulum unfolded protein response.Nat. Rev. Mol. Cell Biol. 2007; 8: 519-529Crossref PubMed Scopus (4851) Google Scholar). Apoptotic pathways are also readied should the adaptive response fail. In addition to intracellular signaling in response to ER stress, there is also extracellular communication via cytokine and chemokine production (14Zhang K. Kaufman R.J. From endoplasmic-reticulum stress to the inflammatory response.Nature. 2008; 454: 455-462Crossref PubMed Scopus (1502) Google Scholar). The ER stress pathway is coupled to inflammatory responses through activation of key signaling networks such as NF-κB and JAK/STAT, leading to the production of cytokines, chemokines, and reactive species (15Kitamura M. Control of NF-κB and inflammation by the unfolded protein response.Int. Rev. Immunol. 2011; 30: 4-15Crossref PubMed Scopus (94) Google Scholar, 16Meares G.P. Liu Y. Rajbhandari R. Qin H. Nozell S.E. Mobley J.A. Corbett J.A. Benveniste E.N. PERK-dependent activation of JAK1 and STAT3 contributes to endoplasmic reticulum stress-induced inflammation.Mol. Cell Biol. 2014; 34: 3911-3925Crossref PubMed Scopus (140) Google Scholar). In chronic diseases, ER stress and inflammation may collaborate to drive sustained inflammation (14Zhang K. Kaufman R.J. From endoplasmic-reticulum stress to the inflammatory response.Nature. 2008; 454: 455-462Crossref PubMed Scopus (1502) Google Scholar). By targeting ER stress signaling, it may be possible to break this cycle. PERK is a particularly attractive target because it is a key player in ER stress-induced inflammation, and disruption of PERK has been shown to be beneficial in mouse models of Alzheimer disease and prion disease (17Ma T. Trinh M.A. Wexler A.J. Bourbon C. Gatti E. Pierre P. Cavener D.R. Klann E. Suppression of eIF2α kinases alleviates Alzheimer's disease-related plasticity and memory deficits.Nat. Neurosci. 2013; 16: 1299-1305Crossref PubMed Scopus (387) Google Scholar, 18Moreno J.A. Halliday M. Molloy C. Radford H. Verity N. Axten J.M. Ortori C.A. Willis A.E. Fischer P.M. Barrett D.A. Mallucci G.R. Oral treatment targeting the unfolded protein response prevents neurodegeneration and clinical disease in prion-infected mice.Sci. Transl. Med. 2013; 5: 206ra138Crossref PubMed Scopus (425) Google Scholar). endoplasmic reticulum unfolded protein response protein kinase R-like endoplasmic reticulum kinase activating transcription factor Oncostatin M quantitative PCR thapsigargin tunicamycin C/EBP homologous protein CCAAT/enhancer-binding protein. In this study, we tested the hypothesis that ER stress-induced inflammation could be selectively regulated without disrupting normal inflammatory responses. We modulated ER stress signaling by disrupting PERK function genetically or pharmacologically with the small-molecule PERK inhibitor GSK2606414 (19Axten J.M. Medina J.R. Feng Y. Shu A. Romeril S.P. Grant S.W. Li W.H. Heerding D.A. Minthorn E. Mencken T. Atkins C. Liu Q. Rabindran S. Kumar R. Hong X. et al.Discovery of 7-methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), a potent and selective first-in-class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK).J. Med. Chem. 2012; 55: 7193-7207Crossref PubMed Scopus (439) Google Scholar) to demonstrate that an intermediate level of PERK activity greatly reduces inflammatory responses without compromising ER stress-induced adaptive responses or normal immunological function. Overall, this study provides in vitro proof of concept for PERK as a selective immunoregulatory target. PERK has emerged as a critical signaling node linking ER stress and inflammation (14Zhang K. Kaufman R.J. From endoplasmic-reticulum stress to the inflammatory response.Nature. 2008; 454: 455-462Crossref PubMed Scopus (1502) Google Scholar, 20Martinon F. Glimcher L.H. Regulation of innate immunity by signaling pathways emerging from the endoplasmic reticulum.Curr. Opin. Immunol. 2011; 23: 35-40Crossref PubMed Scopus (119) Google Scholar). Consistent with previous reports, ER stress drives the expression of inflammatory cytokines and chemokines, including IL-6 and CCL2, and the ER stress-selective transcription factor CHOP (Fig. 1A). To verify the importance of PERK in driving inflammatory responses, we isolated primary astrocytes from wild-type, PERK heterozygous (+/−), and PERK knock-out (−/−) embryos and induced ER stress in these cells. As shown in Fig. 1B, wild-type and PERK+/− cells responded to ER stress with increased phosphorylation of eIF2α and STAT3. However, as expected, eIF2α phosphorylation was reduced in PERK+/− cells and absent in PERK−/− cells. STAT3 phosphorylation was not reduced in PERK+/− cells and was attenuated in PERK−/− cells. Wild-type astrocytes increased IL-6, CCL2, and CCL20 in response to ER stress, and this was reduced in PERK+/− astrocytes and abolished in PERK−/− cells (Fig. 1C). In contrast, PERK haploinsufficiency did not reduce ER stress-induced expression of the prototypical UPR genes CHOP, ATF4, and GRP78. In PERK−/− cells, induction of CHOP and ATF4 was blocked, whereas up-regulation of GRP78, which is ATF6-dependent (21Yoshida H. Haze K. Yanagi H. Yura T. Mori K. Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins: involvement of basic leucine zipper transcription factors.J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1014) Google Scholar), remained unaffected (Fig. 1C). These data indicate that, with partial loss of PERK, cells are still able to mount an adaptive survival response, albeit with reduced inflammatory cytokine and chemokine production. Consistent with this, wild type and PERK+/− cells exposed to ER stress for 24 h showed little apoptosis, as measured by caspase 3 cleavage, whereas PERK−/− had a marked increase in cleaved caspase 3 (Fig. 1D). To validate these findings, we used an additional genetic approach to acutely knock out PERK. PERK floxed (fl/fl) mice (22Zhang P. McGrath B. Li S. Frank A. Zambito F. Reinert J. Gannon M. Ma K. McNaughton K. Cavener D.R. The PERK eukaryotic initiation factor 2 α kinase is required for the development of the skeletal system, postnatal growth, and the function and viability of the pancreas.Mol. Cell Biol. 2002; 22: 3864-3874Crossref PubMed Scopus (501) Google Scholar) were crossed with the global tamoxifen-inducible cre line, CAGG-CreERTM. Astrocytes were isolated from littermates and treated for 24 h with 4-hydroxytamoxifen to delete one or both alleles of PERK. Seventy-two hours after removal of tamoxifen, astrocytes were treated with thapsigargin. As shown in Fig. 2A, PERK expression and eIF2a phosphorylation were modestly reduced in CreERTM-positive PERKfl/wt astrocytes and absent in CreERTM-positive PERKfl/fl cells. Consistent with the previous data, deletion of one PERK allele reduced ER stress-induced IL-6 expression but not CHOP or ATF4. Deletion of both PERK alleles abrogated IL-6, CHOP, and ATF4 expression (Fig. 2B). These data indicate that, although PERK is essential for survival during ER stress, an intermediate level of PERK will suffice. From these data, we hypothesized that it may be possible to target PERK with partial inhibition as a means to control ER stress-induced inflammation without disrupting adaptive responses to ER stress or normal immunological function.FIGURE 2Inducible deletion of PERK regulates ER stress-induced gene expression. A, astrocytes were isolated from PERKfl/fl, PERKfl/wt × CAGG-CreERTM and PERKfl/fl × CAGG-CreERTM littermates. Astrocytes were treated with 4-hydroxytamoxifen (1 μm) for 24 h to delete the floxed PERK alleles in the Cre-expressing cells. Seventy-two hours after removal of the tamoxifen, cells were treated with thaps (1 μm) for the indicated times, followed by immunoblotting. B, astrocytes were isolated and treated with 4-hydroxytamoxifen as in A and then treated with thaps (1 μm) for 4 h, followed by qPCR analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To test this hypothesis, we used the small-molecule inhibitor of PERK GSK2606414. As shown in Fig. 3A, 0.1 μm GSK2606414 partially inhibited, by ∼50%, thapsigargin-induced eIF2α phosphorylation at 30 min, indicating partial inhibition of PERK. By 4 h, the inhibitory effect of GSK2606414 was still apparent, although diminished. This level of inhibition led to a minor (∼20%) decrease in ATF4 protein expression and did not inhibit the low level of CHOP expression (Fig. 3B). However, it was effective at blocking ER stress-induced IL-6, CCL2, and CCL20 but not CHOP, ATF4, and GRP78 mRNA (Fig. 3C), similar to that observed in PERK+/− cells. Only at high concentrations (1 μm) was GSK2606414 able to block CHOP and ATF4 mRNA expression (Fig. 3C), similar to that observed in PERK−/− cells. GSK2606414 also significantly inhibited ER stress-induced IL-6 and CCL2 protein after 24 h, as measured by ELISA (Fig. 3D). At this time point, GSK2606414 was no longer effective at inhibiting eIF2α phosphorylation but, at high concentrations, did prevent ATF4 and CHOP expression (Fig. 3E). To extend these findings, we examined the inhibitory effect of GSK2606414 on ER stress-induced cytokine and chemokine expression by multiplex ELISA. In addition to the significant inhibition of IL-6 and CCL2, we also observed inhibition of ER stress-induced CXCL10, CXCL1, and CXCL2 (Fig. 4). These data demonstrate that modest PERK inhibition can reduce some inflammatory molecules stimulated by ER stress.FIGURE 4PERK-dependent regulation of cytokines and chemokines. Astrocytes were treated with thaps (1 μm) for 24 h in the absence or presence of GSK2606414 (0.1 μm). Cell culture supernatants were collected and analyzed by multiplex ELISA. n = 3, *, p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Not only does ER stress stimulate inflammation, it can also alter the response to other inflammatory signals (16Meares G.P. Liu Y. Rajbhandari R. Qin H. Nozell S.E. Mobley J.A. Corbett J.A. Benveniste E.N. PERK-dependent activation of JAK1 and STAT3 contributes to endoplasmic reticulum stress-induced inflammation.Mol. Cell Biol. 2014; 34: 3911-3925Crossref PubMed Scopus (140) Google Scholar, 23Martinon F. Chen X. Lee A.-H. Glimcher L.H. TLR activation of the transcription factor XBP1 regulates innate immune responses in macrophages.Nat. Immunol. 2010; 11: 411-418Crossref PubMed Scopus (702) Google Scholar, 24Liu Y.-P. Zeng L. Tian A. Bomkamp A. Rivera D. Gutman D. Barber G.N. Olson J.K. Smith J.A. Endoplasmic reticulum stress regulates the innate immunity critical transcription factor IRF3.J. Immunol. 2012; 189: 4630-4639Crossref PubMed Scopus (90) Google Scholar). To test whether PERK inhibition could modulate this effect, astrocytes were treated with the cytokines IL-1β, OSM, or IFN-γ individually or in the presence of thapsigargin. ER stress enhanced the expression of IL-6 in response to IL-1β, OSM, and IFN-γ (Fig. 5A) and, similarly, enhanced the expression of CCL2 in response to IFN-γ and OSM (Fig. 5B). Cytokines did not affect thapsigargin-induced CHOP expression (data not shown). To determine whether the enhanced inflammatory gene expression was sensitive to PERK inhibition, astrocytes were treated with cytokines and thapsigargin in the absence or presence of the PERK inhibitor. GSK2606414 did not affect gene expression induced by cytokines alone but did attenuate the enhancement by ER stress (Fig. 5, A and B). Additionally, ER stress did not enhance, but instead suppressed, IFN-γ-induced TNFα expression, and this was unaffected by PERK inhibition (Fig. 5C). These data indicate that PERK is dispensable in canonical cytokine signaling and that PERK inhibition can block ER stress-induced augmentation of cytokine-mediated gene expression. Our previous work demonstrated that ER stress-induced IL-6 and CCL2 in astrocytes is dependent on JAK1 (16Meares G.P. Liu Y. Rajbhandari R. Qin H. Nozell S.E. Mobley J.A. Corbett J.A. Benveniste E.N. PERK-dependent activation of JAK1 and STAT3 contributes to endoplasmic reticulum stress-induced inflammation.Mol. Cell Biol. 2014; 34: 3911-3925Crossref PubMed Scopus (140) Google Scholar). However, our data showing that deletion of one PERK allele attenuates IL-6 and CCL2 but does not reduce STAT3 phosphorylation suggests that additional mechanisms contribute to the sensitivity of these genes to PERK inhibition. Many of the PERK-mediated effects are dependent on eIF2α phosphorylation and subsequent expression of ATF4 (25Wek R.C. Cavener D.R. Translational control and the unfolded protein response.Antioxid. Redox. Signal. 2007; 9: 2357-2371Crossref PubMed Scopus (234) Google Scholar). Therefore, we tested whether the eIF2α-induced translational inhibition was involved in the regulation of these inflammatory genes using the recently described compound ISRIB (26Sidrauski C. Acosta-Alvear D. Khoutorsky A. Vedantham P. Hearn B.R. Li H. Gamache K. Gallagher C.M. Ang K.K. Wilson C. Okreglak V. Ashkenazi A. Hann B. Nader K. Arkin M.R. et al.Pharmacological brake-release of mRNA translation enhances cognitive memory.eLife. 2013; 2: e00498Crossref PubMed Scopus (397) Google Scholar). This compound reverses the translational block brought on by phosphorylation of eIF2α through an agonistic effect on EIF2B (27Sidrauski C. Tsai J.C. Kampmann M. Hearn B.R. Vedantham P. Jaishankar P. Sokabe M. Mendez A.S. Newton B.W. Tang E.L. Verschueren E. Johnson J.R. Krogan N.J. Fraser C.S. Weissman J.S. et al.Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response.eLife. 2015; 4: e07314Crossref PubMed Scopus (165) Google Scholar). As expected, ISRIB suppressed thapsigargin-induced ATF4 protein expression, had no effect on ATF4 mRNA, and modestly reduced CHOP mRNA (Fig. 5A). Interestingly, this highlights that PERK controls ATF4 at two levels through distinct mechanisms. ATF4 protein is dependent on eIF2α-induced translational inhibition, whereas ATF4 mRNA is PERK-dependent but independent of eIF2α. These data are consistent with data showing that PERK-NRF2 signaling drives ATF4 transcription (28Afonyushkin T. Oskolkova O.V. Philippova M. Resink T.J. Erne P. Binder B.R. Bochkov V.N. Oxidized phospholipids regulate expression of ATF4 and VEGF in endothelial cells via NRF2-dependent mechanism: novel point of convergence between electrophilic and unfolded protein stress pathways.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 1007-1013Crossref PubMed Scopus (115) Google Scholar). Concomitant with suppression of ATF4 protein, ISRIB potently suppressed thapsigargin-induced IL-6, CCL2, and CCL20 expression (Fig. 6B). This suggests that ER stress-induced inhibition of translation promotes transcription of inflammatory molecules such as IL-6, CCL2, and CCL20. Importantly ISRIB does not broadly block inflammatory gene expression, as it had no effect on cytokine-induced IL-6 or CCL2 expression (data not shown). To test whether ATF4 connects translational repression to inflammatory gene expression, we used siRNA to knock down ATF4. As shown in Fig. 6C, ATF4 expression is reduced, and thapsigargin-induced expression of the ATF4 target gene tribbles 3 (Trib3) (29Han J. Back S.H. Hur J. Lin Y.-H. Gildersleeve R. Shan J. Yuan C.L. Krokowski D. Wang S. Hatzoglou M. Kilberg M.S. Sartor M.A. Kaufman R.J. ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death.Nat. Cell Biol. 2013; 15: 481-490Crossref PubMed Scopus (1048) Google Scholar) is abrogated by ATF4 siRNA. This confirms functional silencing of ATF4. Knockdown of ATF4 did not inhibit thapsigargin-induced IL-6 and partially reduced CCL2 expression (Fig. 6C). Collectively, these data indicate that the eIF2α-induced translational block is essential for inflammatory gene expression but is largely independent of ATF4. We previously demonstrated that PERK-dependent communication between ER-stressed astrocytes and naïve microglia resulted in the activation of microglia (16Meares G.P. Liu Y. Rajbhandari R. Qin H. Nozell S.E. Mobley J.A. Corbett J.A. Benveniste E.N. PERK-dependent activation of JAK1 and STAT3 contributes to endoplasmic reticulum stress-induced inflammation.Mol. Cell Biol. 2014; 34: 3911-3925Crossref PubMed Scopus (140) Google Scholar). Therefore, we tested whether PERK inhibition could also modulate astrocyte-microglia interactions. Astrocytes were treated transiently (2 h) with thapsigargin in the absence or presence of GSK2606414, washed thoroughly to remove the thapsigargin, and cultured in fresh medium with or without GSK2606414. Following the addition of fresh medium, primary microglia were added in a Transwell (Fig. 7A). The microglia were cultured with astrocytes for 24 h. As shown in Fig. 7B, astrocytes under ER stress promoted a proinflammatory M1-like phenotype in microglia that included down-regulation of the M2 markers arginase, CD206, and IGF1 and up-regulation of inflammatory genes, including IL-1β, TNFα, inducible nitric-oxide synthase (iNOS), and IL-12p40. PERK inhibition had no effect on the down-regulation of M2 markers but did reduce the levels of IL-1β, TNFα, and inducible nitric-oxide synthase. The expression of IL-12p40 was unaffected by PERK inhibition (Fig. 7B). These data indicate that ER-stressed astrocytes promote a proinflammatory phenotype in microglia and that this can be attenuated by inhibiting PERK. A major challenge in managing inflammation is to suppress chronic, pathological inflammation while leaving normal beneficial immunological functions intact. Our observation that PERK inhibition suppresses ER stress-induced inflammatory gene expression but not cytokine-induced gene expression (Fig. 5) suggests that targeting PERK may not disrupt normal immune function. To test this, we used several primary cell types to assess the immunological impact of PERK inhibition. Macrophage-mediated phagocytosis is a key component in innate immunity. Treatment of bone marrow-derived macrophages for 24 h with GSK2606414 did not cause toxicity (data not shown) or impair phagocytosis (Fig. 8A). Next, we examined the inflammatory response in primary microglia to the bacterial component LPS. Similar to our findings with cytokines, LPS stimulated potent inflammatory gene expression that was not affected by PERK inhibition (Fig. 8B). Differentiation of naïve CD4+ T cells into various effector T cell subsets is paramount in establishing an appropriate adaptive immune response. As such, we tested whether PERK inhibition could alter CD4+ T cell polarization. T cells were cultured under Th1- or Th17-polarizing conditions in the absence or presence of GSK2606414 for 3 days. As shown in Fig. 8C, GSK2606414 had little effect on Th1 or Th17 polarization, as assessed by their hallmark cytokines IFN-γ and IL-17A, respectively. These data indicate that, although PERK inhibition can suppress inflammation under ER stress conditions, targeting PERK does not have a broad impact on overall immunological function. In line with these results, PERK has also been shown to be dispensable for B cell development and immunoglobulin secretion (30Gass J.N. Jiang H.-Y. Wek R.C. Brewer J.W. The unfolded protein response of B-lymphocytes: PERK-independent development of antibody-secreting cells.Mol. Immunol. 2008; 45: 1035-1043Crossref PubMed Scopus (104) Google Scholar). Neurological diseases are a growing public health problem. Decades of research have greatly expanded our knowledge of neuroscience but have yet to lead to effective therapies, largely because of the complex nature of these diseases. We now appreciate that critical non-neuronal factors, such as neuroinflammation, are involved in neurological diseases. Specifically, inflammation is thought to contribute to the chronic non-resolving pathology common to neurodegenerative diseases (5Ransohoff R.M. Brown M.A. Innate immunity in the central nervous system.J. Clin. Invest. 2012; 122: 1164-1171Crossref PubMed Scopus (703) Google Scholar). Selectively disrupting disease-driven inflammation while leaving normal immune function intact could provide a new therapeutic avenue. Unfortunately, targets to achieve such specificity are currently unknown. In this study, we provide evidence that small molecule inhibition or genetic haploinsufficiency of the UPR kinase PERK can selectively control inflammation brought on by ER stress without impinging on UPR-dependent survival and adaptive responses. In healthy tissue, an inflammatory response is" @default.
• W2492545900 created "2016-08-23" @default.
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• W2492545900 date "2016-07-01" @default.
• W2492545900 modified "2023-10-15" @default.
• W2492545900 title "Attenuation of PKR-like ER Kinase (PERK) Signaling Selectively Controls Endoplasmic Reticulum Stress-induced Inflammation Without Compromising Immunological Responses" @default.
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