Interleukin-1β-induced inflammatory signaling in C20 human microglial cells

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1 Dvis et l. Neuroimmunol Neuroinflmmtion 2018;5:50 DOI: / Neuroimmunology nd Neuroinflmmtion Originl Article Open Access Interleukin-1β-induced inflmmtory signling in C20 humn microglil cells Rndll L. Dvis 1, Dniel J. Buck 1, Kelly McCrcken 1, Gry W. Cox 1, Suhs Ds 2 1 Deprtment of Phrmcology & Physiology, Oklhom Stte University Center for Helth Sciences, Tuls, OK 74107, USA. 2 Deprtment of Antomy & Cell Biology, Oklhom Stte University Center for Helth Sciences, Tuls, OK 74107, USA. Correspondence to: Dr. Rndll L. Dvis, Dniel J. Buck, Kelly McCrcken nd Gry W. Cox, Deprtment of Phrmcology & Physiology, Oklhom Stte University Center for Helth Sciences, 1111 W. 17th Street, Tuls, OK 74107, USA. E-mils: rndll. dvis@okstte.edu; dniel.uck@okstte.edu; kelly.mccrcken@okstte.edu; gry.cox@okstte.edu; Dr. Suhs Ds, Deprtment of Antomy & Cell Biology, Oklhom Stte University Center for Helth Sciences, 1111 W. 17th Street, Tuls, OK 74107, USA. E-mil: suhs.ds@okstte.edu How to cite this rticle: Dvis RL, Buck DJ, McCrcken K, Cox GW, Ds S. Interleukin-1β-induced inflmmtory signling in C20 humn microglil cells. Neuroimmunol Neuroinflmmtion 2018;5:50. Received: 26 Sep 2018 First Decision: 1 Nov 2018 Revised: 29 Nov 2018 Accepted: 29 Nov 2018 Pulished: 26 Dec 2018 Science Editor: Athnssios P. Kyritsis Copy Editor: Ci-Hong Wng Production Editor: Hun-Ling Wu Astrct Aim: Incresed inflmmtory signling in microgli is implicted in the pthogenesis of neurodegenertive diseses, trum, psychitric disorders, nd nxiety/depression. Understnding inflmmtory signling in microgli is criticl for dvncing tretment options. Studying rodent-derived microgli hs yielded sustntil informtion, yet, much remins to etter understnd inflmmtory signling in humn microgli. Hence, there is gret interest in developing immortlized humn microglil cell lines. The C20 humn microglil cell line ws recently developed nd our primry ojective ws to dvnce our knowledge of inflmmtory signling in these cells. Methods: Expression of the microgli specific mrker trnsmemrne protein 119 (TMEM119) ws ssessed y western lot nlysis. Lipopolyscchride (LPS)- nd interleukin-1β (IL-1β)-induced cytokine/chemokine expression ws determined y ELISA. Phosphoryltion of inhiitory kpp B lph (IκBα), nucler fctor (NF)-κB p65, nd p38 mitogen-ctivted protein kinse (p38 MAPK) ws mesured y western lot nlysis. Results: TMEM119 ws expressed in unstimulted C20 cells, nd to greter extent in IL-1β-stimulted cells. IL-1β significntly induced IL-6, monocyte chemottrctnt protein-1/ccl2, nd interferon-γ inducile protein 10/CXCL10 expression. LPS induced CCL2 expression, ut not IL-6 or CXCL10 expression. IL-1β induced inflmmtory signling s indicted y incresed phosphoryltion of IκBα, NF-κB p65 nd p38 MAPK. Conclusion: We provide the first evidence tht C20 microgli express TMEM119. This is the initil report of IL-1βinduced ctivtion of IκBα, NF-κB p65, nd p38 MAPK nd susequent CXCL10, CCL2 nd IL-6 secretion in C20 The Author(s) Open Access This rticle is licensed under Cretive Commons Attriution 4.0 Interntionl License ( which permits unrestricted use, shring, dpttion, distriution nd reproduction in ny medium or formt, for ny purpose, even commercilly, s long s you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde.

2 Pge 2 of 11 Dvis et l. Neuroimmunol Neuroinflmmtion 2018;5:50 I cells. These findings dvnce our understnding of inflmmtory signling in C20 cells nd support the vlue of this cell line s reserch tool. Keywords: Interleukin-1β, chemokine, microgli, p38, nucler fctor-κb p65, neuroinflmmtion INTRODUCTION Microgli re resident mcrophges in the centrl nervous system (CNS) nd re essentil to rin physiology; ut lso, they re instrumentl in response to injury nd infection in the CNS (See Wolf et. l. [1] for review). Microgli constntly survey their locl environment nd respond to extrcellulr cues (e.g., ATP) to mintin homeostsis [2,3]. Other specific physiologicl functions of microgli include removl of ded neurons nd cellulr deris [2,3], synptic pruning [4], nd regultion of synptic connectivity nd plsticity [5-7]. Microgli re lso integrl to innte immunity nd re instrumentl in neuroinflmmtion [8,9]. For instnce, ctivted microgli relese of pro-inflmmtory meditors including, cytokines [e.g., interleukin (IL)- 1β, IL-6] [10,11], chemokines (e.g., monocyte chemottrctnt protein-1/ccl2, interferon-γ inducile protein 10/CXCL10) [11-13] nd rective oxygen species [14,15]. Microgli lso modulte the inflmmtory response y relesing nti-inflmmtory cytokines such s IL-10 [16] nd trnsforming growth fctor (TGF)-β [17]. Controlled neuroinflmmtion is neuroprotective [18], however, excessive or chronic neuroinflmmtion is neurotoxic, it contriutes to neurodegenertion, nd disrupts neuronl function [1]. For instnce, microglil ctivtion nd neuroinflmmtion re present in neurodegenertive diseses, CNS infection nd trum, s well s psychitric disorders. Indeed, emerging evidence suggests phrmcologicl modultion of microgli my e eneficil in treting certin CNS disorders [19-22]. Much hs een discovered out microgli function, including inflmmtory signling, using in vitro pproches with primry cell cultures nd trnsformed cell lines [23-25]. Significnt insights hve een otined out inflmmtory signling using either primry rt or mouse microgli [26-29] or trnsformed cell lines such s BV-2 murine microglil cells [26,28-30]. While mny of the findings hve een oserved in primry humn microgli [14,31,32], not surprisingly, there hve een differences oserved [24]. For exmple, cteril lipopolyscchride (LPS) nd interferon-γ (IFNγ) re potent inducers of inflmmtory signling (e.g., cytokine/chemokine expression) in oth mouse nd humn microgli [29,32-34], wheres, IL-1β ctivtes humn, ut not mouse microgli [10,11,35,36]. The lck of responsiveness of mouse microgli to IL-1β stimultion is likely consequence of very low IL-1 receptor (IL-1R) expression [36]. Therefore, while there hs een welth of knowledge otined out rodent-derived microgli, it remins criticl to etter understnd inflmmtory signling in humn microgli. Primry humn microgli (fetl nd dult) remin necessry tool, ut they re reltively difficult nd expensive to otin nd use, thus, only limited numer of reserch groups hve ccess, funds, nd expertise to extensively use this pproch. Primry humn microgli re commercilly ville (e.g., ScienCell Reserch Lortories, ct. #HM- 1900) however, they re often in limited supply or not in stock. Therefore, there is incresing interest in immortlized humn microglil cell lines s sustinle tools for studying microgli function [37-39]. The importnce of humn microglil cell lines extends eyond sic understnding of microgli function ut lso these cell lines serve s pltform for investigting the effects of phrmcologic gents on microgli, with n eye towrd drug development. Recently, novel, immortlized humn microglil cell line (C20) ws introduced to the field; the cells mintin microglil morphology, express multiple cell surfce microgli mrkers, nd express proinflmmtory cytokines following stimultion with tumor necrosis fctor-α (TNFα) [37]. We re prticulrly interested in IL-1β-induced inflmmtory signling in microgli due to the estlished role of IL-1β in neuroinflmmtion [40-43]. Interestingly, most studies utilize LPS nd/or IFNγ to stimulte microgli

3 Dvis et l. Neuroimmunol Neuroinflmmtion 2018;5:50 I Pge 3 of 11 in vitro; nd to lrge extent this is ecuse most in vitro studies utilize rodent cells nd these re the stimuli to which they respond. Therefore, the primry ojective of our investigtion ws to dvnce our knowledge of IL-1β-induced inflmmtory signling in humn microgli using C20 humn microglil cells. METHODS Cells C20 humn microglil cells were otined from Dvid Alvrez-Cronell, PhD (Cse Western Reserve University) nd detils pertining to the genertion of this cell line were recently reported [37]. Briefly, these investigtors otined humn microgli from ScienCell Reserch Lortories, Crlsd, CA (Ct# HM1900) nd then immortlized the cells using simin virus 40 lrge T ntigen nd htert (to fcilitte expression of humn telomerse reverse trnscriptse) [37]. The C20 cells tht we otined were confirmed to e of humn origin y the Humn Identity Testing Lortory t Oklhom Stte University Center for Helth Sciences, which utilized the PowerPlex 21 System (Promeg, Mdison, WI), multiplex short tndem repet system for humn identifiction, s previously descried [44]. For our experiments, cells were used t pssges 5-10 nd were either seeded in 24-well pltes ( cells/well) or in 100 mm dishes ( cells) depending on the experiment nd cultured in growth medium [Dulecco s Modified Egle Medium/Hm s F-12 50/50 mix supplemented with 2.5 mmol/l L-glutmine (Corning CV), 10% fetl ovine serum (Atlnt Biologicls S11550), nd 1% penicillin/streptomycin (Lonz 17603E)] until 90% confluent (4-5 dys). Medium ws replced with serum-free medium (SFM) 24 h prior to stimultion. Norml humn strocytes (NHA, ScienCell, #HA1800) were mintined s previously descried [45]. Stimulus C20 were stimulted in SFM contining either LPS (E. coli K12, 1 µg/ml; InvivoGen, Sn Diego, CA) or humn recominnt IL-1β (20 ng/ml; Peprotech, Rocky Hill, NJ) for 10 min - 24 h depending on the specific experiment; wheres, NHA were stimulted with IL-1β (3 ng/ml) for 24 h in the single study in which they were used. Detils regrding the numer of independent experiments nd replicte tretments within ech experimentl run re provided in the figure legends. Expression of microglil mrker While the precise function of trnsmemrne protein 119 (TMEM119) hs yet to e determined, it is incresingly recognized s relile mrker of humn microgli tht discrimintes microgli in the rin from lood-derived mcrophges [46-49]. We ssessed TMEM119 expression y western lot nlysis nd fluorescent immunocytochemistry. For western lot nlysis, whole cell lystes were collected from unstimulted nd IL-1β-stimulted C20 cells (cultured in 100 mm dishes), using Triton X-100 lysis uffer (50 mmol/l Tris-HCl, 150 mmol/l NCl, 10% glycerol, 1% Triton X-100) contining MS-SAFE protese/ phosphtse inhiitor (Sigm-Aldrich). Briefly, cells were rinsed with cold phosphte uffered sline (PBS), then lysed in 300 µl of lysis uffer nd collected into 1.5 ml tues. The lystes were then incuted on ice for 45 min with intermittent mixing y inversion. Lystes were centrifuged for 10 min t 20,800 g nd 4 C. The superntnt, contining whole cell protein ws then collected nd stored t -80 C. Thirty microgrms of totl protein were loded on 7.5% polycrylmide gels (BioRd TGX FstCst Acrylmide kit, # ), electrophoresed nd then trnsferred to polyvinylidene difluoride (PVDF) memrne. Memrnes were then incuted t 4 C for h with rocking. Memrnes were locked with 5% ovine serum lumin (BSA) for 2 h prior to incution with ntiodies. Primry ntiodies included, nti-tmem119 (1:100, Sigm #HPA051870), nti-glil firillry cidic protein (GFAP; 1:5000, Millipore #MAB360), nd nti-β-tuulin (1:1000, Cell Signling #2146S). Anti-rit IgG, AP-linked (1:1000, Cell Signling #7054S) ws used s the secondry ntiody. Whole cell lystes from NHA were used for comprison. Restore western lot stripping uffer (Thermo Scientific #21059) ws used to remove ntiodies nd llow for re-lelling of memrnes. The lots were scnned in phosphoimger Typhoon 9410 (GE Helthcre, Uppsl, Sweden) using Amershm ECF Sustrte #RPN-5785, nd Imge J (Ntionl Institutes of Helth) ws used for densitometric nlysis.

4 Pge 4 of 11 Dvis et l. Neuroimmunol Neuroinflmmtion 2018;5:50 I TMEM119 expression ws lso ssessed y fluorescent immunocytochemistry. Briefly, C20 cells were seeded on glss coverslips in 6-well dishes nd cultured s descried ove until 70%-80% confluent. Following the tretment period (24 h), cells were wshed three times with PBS then fixed in 4% prformldehyde for 15 min. Cells were wshed with PBS nd then incuted in locking uffer (1% BSA in PBS) overnight t room temperture. Cells were then incuted in nti-tmem119 (1:500 in PBS contining 0.5% BSA, Sigm #HPA051870) overnight t 4 C with rocking. After wshing with PBS, cells were incuted for 2 h in Alex Fluor 488 donkey nti-rit ntiody (1:3000 in PBS contining 0.5% BSA; Life Technologies #A-21206). Cells were wshed gin with PBS, treted with 300 nmol/l 4,6-dimidino-2-phenylindole (DAPI; Sigm) without rocking for 15 min t room temperture, then wshed in PBS prior to mounting in Prolong Gold nti-fde regent (Invitrogen; Eugene, OR). Cytokine/chemokine expression Levels of secreted CXCL10, CCL2, nd IL-6 were mesured in the culture medium using stndrd dulntiody solid phse immunossy (ELISA) kits purchsed from Peprotech. Cytokine/chemokine concentrtions were normlized to totl cellulr protein levels, which were determined using the icinchoninic cid protein ssy s previously descried [50]. Expression of inflmmtory signling molecules Induction of inflmmtory signling ws determined y mesuring phosphoryltion of inhiitory kpp B lph (IκBα), nd p38 mitogen-ctivted protein kinse (p38 MAPK) in cytoplsmic frctions; nd phosphoryltion of nucler fctor (NF)-κB p65 in nucler frctions. More specificlly, fter experimentl tretments, cells in 100 mm dishes were wshed twice with PBS nd nucler nd cytoplsmic protein extrcts prepred s previously descried [51], with the exception of including 1 mmol/l sodium orthovndte (Sigm, #450243) in the lysis uffers. Thirty microgrms of totl protein were loded on 7.5% polycrylmide gels (BioRd TGX FstCst Acrylmide kit, # ), electrophoresed nd then trnsferred to PVDF memrne. Memrnes were then incuted t 4 C for h with rocking. Primry ntiodies included nti-phospho-iκbα (1:100, Cell Signling #2859S), nti-iκbα (1:1000, Cell Signling #4812S), ntiphospho-p38 (1:500, Cell Signling #9215S), nti-p38 (1:1000, Cell Signling #9212S), nti-phospho-nfκb p65 (1:1000, Cell Signling #3033S), nti-nf-κb p65 (1:1000, Cell Signling #4764S), nd nti-β-tuulin (1:1000, Cell Signling #2146S). Anti-rit IgG, AP-linked (1:1000, Cell Signling #7054S) ws used s the secondry ntiody nd restore western lot stripping uffer ws used to remove ntiodies nd llow for relelling of memrnes. The lots were scnned nd nlyzed s descried ove in the section on microgli mrker expression. Sttisticl nlyses Prism version 7.0 softwre (GrphPd Inc., Sn Diego, CA) ws used for figure presenttion nd sttisticl nlysis. Anlyses included one-wy nlysis of vrince (ANOVA), followed y Tukey s multiple comprisons test. In those instnces where dt did not exhiit homogeneity of vrince (P < 0.05 for Brtlett s test), dt were log-trnsformed prior to nlysis. Dt re presented s men ± SEM (n = 4-7) nd P < 0.05 indicted sttisticlly significnt differences etween groups. RESULTS Expression of microglil mrker TMEM119 ws roustly expressed in IL-1β-treted C20 cells s indicted y strong nd t 29 kd [Figure 1 Top]. Wheres, TMEM119 ws not detected in either unstimulted C20 cells or in NHA y western lot nlysis. As expected, the strocyte mrker, GFAP ws expressed in NHA, ut ws not detected in C20 cells. Using fluorescent immunocytochemistry we confirmed our western lot findings tht IL-1β-treted C20 cells express TMEM119 [Figure 1 Bottom]. This immunocytochemistry pproch reveled tht TMEM119 is lso expressed in unstimulted C20 cells. In oth unstimulted nd IL-1β-stimulted cells, TMEM119 ws

5 Dvis et l. Neuroimmunol Neuroinflmmtion 2018;5:50 I Pge 5 of 11 C20 NHA TMEM119 -tuulin GFAP -tuulin US US 29 kd 55 kd 51 kd 55 kd C20 A B C D E F G H Figure 1. C20 humn microglil cells express the microglil mrker trnsmemrne protein 119 (TMEM119). C20 cells were exposed to medi lone (unstimulted; US) or medi contining interleukin-1β (IL-1β) (20 ng/ml) for 24 h. Top pnel: western lot nlysis ws used to mesure levels of TMEM119 nd glil firillry cidic protein (GFAP) in whole cell lystes nd β-tuulin ws ssessed s loding control. Whole cell lystes from unstimulted nd IL-1β (3 ng/ml)-stimulted norml humn strocytes (NHA) were used for comprison. The lots presented re representtive of independent experiments (n = 3 for C20; nd n = 2 for NHA). Bottom pnel: fluorescent immunocytochemistry ws used to further ssess TMEM119 expression (green) in US (A-D) nd IL-1β-stimulted (E-H) C20 cells; nd nuclei were leled with 4,6-dimidino-2-phenylindole (DAPI, lue). Imges re shown t 400 mgnifiction. The rrows in ox H highlight the cytoplsmic extensions detected predominntly in the cytoplsmic nd/or cell memrne regions [Figure 1 Bottom]. Consistent with the western lot findings, TMEM119 expression ws more pronounced in the IL-1β-treted C20 cells compred to unstimulted cells. Cytokine/chemokine expression C20 cells constitutively expressed only miniml mounts of CXCL10, CCL2, nd IL-6 [Figure 2]. However, stimultion with IL-1β significntly (P < ) induced expression of CXCL10, CCL2, nd IL-6. In contrst to IL-1β, LPS induced only miniml, yet significnt (P < 0.01), increse in CCL2 expression, nd did not significntly (P > 0.05) ffect CXCL10 or IL-6 expression. Expression of inflmmtory signling molecules Levels of phosphorylted IκBα in the cytoplsm were significntly (P < ) incresed fter 10 min IL- 1β exposure, remined elevted out to 60 min, ut were eginning to drop towrd seline levels [Figure 3]. IκBα ws constitutively expressed, with levels significntly (P < ) reduced 10 min fter IL-1β tretment. IκBα expression remined significntly (P < 0.001) reduced 30 min fter stimultion, ut incresed ck to seline levels y 60 min.

6 Pge 6 of 11 Dvis et l. Neuroimmunol Neuroinflmmtion 2018;5:50 I CXCL10 (pg/mg protein) 8,000 7,000 6,000 5,000 4,000 3,000 2,000 1, LPS CCL2 (pg/mg protein) 600, , , , , ,000 20,000 0 LPS c IL-6 (pg/mg protein) 400, , , , , , ,000 50,000 1,000 0 LPS Figure 2. Cytokine/chemokine expression in C20 humn microglil cells. C20 cells were exposed to medi lone (unstimulted; ) or medi contining either lipopolyscchride (LPS) (1 µg/ml) or interleukin-1β (IL-1β) (20 ng/ml) for 24 h. CXCL10, CCL2, nd IL-6 levels in the culture medium were determined y ELISA nd normlized to totl cellulr protein (s determined y the icinchoninic cid method). Dt represent men ± SEM (n = 4-7). Brs with different letters re significntly different (P < 0.01) s determined y one-wy ANOVA nd Tukey s pirwise comprisons p-ikb IkB -tuulin US 40 kd 39 kd 55 kd p-ikb/ikb (reltive density) p-ikb/-tuulin (reltive density) Figure 3. Interleukin-1β (IL-1β)-induced inhiitory kpp B lph (IκBα) ctivtion in C20 humn microglil cells. C20 cells were exposed to medi lone (unstimulted; US) or medi contining IL-1β (20 ng/ml) for min. Western lot nlysis ws used to mesure levels of p-iκbα, IκBα, nd β-tuulin in cytoplsmic protein extrcts. The lots presented re representtive of independent experiments (n = 4-5) nd the dt represent men ± SEM. Brs with different letters re significntly different (P < 0.05) s determined y one-wy ANOVA nd Tukey s pirwise comprisons The expression of phospho-p65 in the nucleus rpidly incresed within 10 min in response to IL-1β tretment (P < ), efore declining to seline levels y 30 min [Figure 4]. Constitutive expression of p65 in the nucleus of C20 cells ws evident nd levels remined unchnged (P = 0.37) throughout the 60 min exposure to IL-1β. Phosphoryltion of p38 in the cytoplsm occurred rpidly following tretment with IL-1β s indicted y significnt (P < ) increse in phospho-p38/p38 fter 10 min [Figure 5]. The levels of phospho-p38 then rpidly declined y 30 min (P < 0.05), nd returned to seline y 60 min (P = 0.19). Constitutive expression of p38 in the cytoplsm of C20 cells ws roust nd the expression levels remined constnt (P = 0.58) throughout the 60 min exposure to IL-1β. DISCUSSION Microgli re key component of the innte immune system with criticl roles in response to injury nd infection [1]. Furthermore, microgli re instrumentl in neurodevelopment nd physiologicl functions necessry for mintining CNS homeostsis [52]. Microgli hve een implicted in multitude of CNS disorders, nd modultion of microgli function hs emerged s potentil therpeutic strtegy [8].

7 Dvis et l. Neuroimmunol Neuroinflmmtion 2018;5:50 I Pge 7 of 11 p-p65 p65 -tuulin US 65 kd 65 kd 55 kd p-p65/p65 (reltive density) p65/-tuulin (reltive density) P = Figure 4. Interleukin-1β (IL-1β)-induced nucler fctor (NF)-κB p65 ctivtion in C20 humn microglil cells. C20 cells were exposed to medi lone (unstimulted; US) or medi contining IL-1β (20 ng/ml) for min. Western lot nlysis ws used to mesure levels of p-nf-κb p65, NF-κB p65, nd β-tuulin in nucler protein extrcts. The lots presented re representtive of independent experiments (n = 4-5) nd the dt represent men ± SEM. Brs with different letters re significntly different (P < 0.05) s determined y one-wy ANOVA nd Tukey s pirwise comprisons p-p38 p38 -tuulin US 43 kd 43 kd 55 kd p-p38/p38 (reltive density) P = c 0.5 c 0.0 p38/-tuulin (reltive density) Figure 5. Interleukin-1β (IL-1β)-induced p38 MAPK ctivtion in C20 humn microglil cells. C20 cells were exposed to medi lone (unstimulted; US) or medi contining IL-1β (20 ng/ml) for min. Western lot nlysis ws used to mesure levels of p-p38 mitogen-ctivted protein kinse (p38 MAPK), p38 MAPK, nd β-tuulin in cytoplsmic protein extrcts. The lots presented re representtive of independent experiments (n = 5) nd the dt represent men ± SEM. Brs with different letters re significntly different (P < 0.05) s determined y one-wy ANOVA nd Tukey s pirwise comprisons Altogether, there is sustntil interest in further understnding microgli function. Much of wht is currently known out microgli, stems from in vitro studies using rodent microgli, prticulrly immortlized cell lines. However, there is incresing interest in the use of immortlized humn microglil cell lines to dvnce our understnding of humn microgli function nd discovery of phrmcologicl gents tht modulte microgli [37,38,53,54]. Among the very few immortlized humn microglil cell lines ville for use is the C20 humn microglil cell line recently developed y Grci-Mes et l. [37]. These investigtors utilized RNA sequencing to confirm the microglil phenotype of C20 cells nd they demonstrted tht these cells mintin migrtory cpcity nd phgocytic ctivity chrcteristic of microgli [37]. Furthermore, C20 cells express numerous microglil surfce mrkers, including cluster of differentition (CD)11, CD68, TGFβ receptor (TGFβR), nd the P2 purinergic receptor, P2RY12, s indicted y immunofluorescence nd flow cytometry [37]. While CD11 + mcrophges re lso present in peripherl tissues [55], TGFβR nd P2RY12 hve een suggested to e microglil specific [56,57]. TMEM119 hs lso recently emerged s microgli mrker cple of discriminting etween resident microgli nd peripherl mcrophges [46,47], yet the functionl importnce of this protein remins to e elucidted. We hve demonstrted for the first time tht C20 cells express TMEM119. Interestingly, TMEM119 protein expression ws gretest in C20 cells tht were stimulted with IL-1β. While we did detect very smll mounts of TMEM119 in unstimulted C20 cells during preliminry experiments, sustntil mount of totl protein hd to e loded into the gels in order to chieve these

8 Pge 8 of 11 Dvis et l. Neuroimmunol Neuroinflmmtion 2018;5:50 I results. Wheres, when 30 µg of protein from unstimulted C20 cells were electrophoresed, we did not detect TMEM119. Further investigtion is wrrnted to fully pprecite the expression profile of TMEM119 t the mrna nd protein levels; nd the modultory influence of proinflmmtory meditors. Additionl insights into the expression profile nd functionl role of TMEM119 in microgli re therefore needed nd C20 cells my prove to e useful tool in this line of investigtion. Previously, during the initil chrcteriztion of this cell line, C20 cells were found to secrete proinflmmtory cytokines following stimultion with TNFα [37]. We hve dded to these findings nd dvnced our understnding of inflmmtory signling in C20 cells. More specificlly, we provide the first evidence tht IL-1β potently induces expression of CXCL10, CCL2, nd IL-6 in C20 cells. We hve lso demonstrted tht LPS differentilly ffects expression of these inflmmtory meditors s evidenced y stimultion of CCL2 expression, ut not CXCL10 or IL-6. Overll, it is cler tht IL-1β is much more effective inducer of cytokine/ chemokine expression in C20 cells compred to LPS. In contrst, mouse microgli (including the BV-2 cells) re very responsive to LPS, ut not to IL-1β due to the sence of IL-1R expression [36]. IL-1β levels re elevted in rnge of CNS disorders nd this proinflmmtory cytokine hs een implicted s key meditor of neuropthology [40]. Thus, it is fundmentlly importnt to fully understnd IL-1βinduced inflmmtory signling in humn microgli. Furthermore, dvncing our understnding of these signling events in C20 humn microgli is criticl for developing this reserch tool. Secretion of CXCL10, CCL2 nd IL-6 y C20 microgli in response to IL-1β exposure is functionlly relevnt endpoint mesure given oth the importnt neurophysiologicl nd neuropthologicl roles of these cytokines/chemokines. For instnce, CXCL10 is initilly neuroprotective in virl infections [58], ut cn lso contriute to neuropthology s evidenced in humn immunodeficiency virus (HIV)-induced dementi [59,60]. CXCL10 lso plys role in neuropthology ssocited with trumtic rin injury (TBI) [61], nd emerging dt suggest CXCL10 is involved in sickness ehvior [62]. CCL2 functions s chemotctic cytokine, ctivting nd directing migrtion of numerous cell types [61] ; nd it is incresingly evident tht incresed levels of this chemokine in the rin contriute to neuropthology of HIV-dementi, AD, ischemi, epilepsy, nd TBI [61]. The cytokine IL-6 cts in the hypothlmus s regultor of metolism [63] nd hs gined ttention for its involvement in utism [64], mjor depression [65,66], nd neurodegenertive diseses [67-69]. Therefore, C20 microgli re expected to e useful tool in discovery of phrmcologic gents tht my modulte microglil ctivtion nd susequent relese of proinflmmtory fctors. We lso provide the first evidence tht IL-1β induces ctivtion of key proinflmmtory signling molecules in C20 cells, including IκBα, NF-κB p65, nd p38 MAPK. The importnce of these signling molecules in microgli ctivtion is well estlished nd these proteins re vile trgets for phrmcologic modultion of inflmmtion [29,34,70-73]. Therefore, y demonstrting tht these signling pthwys re functionl in C20 cells, it is expected tht these cells will e instrumentl in the identifiction nd chrcteriztion of novel phrmcologic gents intended to lter microglil function. In conclusion, we hve determined tht IL-1β-ctivted C20 microgli express the microgli specific mrker TMEM119. Additionlly, we hve provided the first evidence tht IL-1β induces ctivtion of IκBα, NF-κB p65, nd p38 MAPK nd susequent secretion of CXCL10, CCL2 nd IL-6 in C20 humn microgli. These findings support the use of this humn microglil cell line s reserch tool to dvnce our understnding of microgli function nd for the development of phrmcotherpies trgeting rnge of neuropthologies. DECLARATIONS Acknowledgements We gretly pprecite the technicl ssistnce of Roert W. Allen, PhD nd Jun Fu, PhD t the Humn Identity Testing Lortory in the School of Forensic Sciences t Oklhom Stte University Center for Helth Sciences.

9 Dvis et l. Neuroimmunol Neuroinflmmtion 2018;5:50 I Pge 9 of 11 Authors contriutions Concept, experimentl design, literture review, sttisticl nlysis, mnuscript preprtion: Dvis RL Performed experiments, dt cquisition nd nlysis, nd mnuscript editing: Buck DJ Performed experiments, experimentl design, dt cquisition nd nlysis, nd mnuscript editing: McCrcken K Performed experiments, dt cquisition, nd mnuscript editing: Cox GW Performed experiments, dt cquisition nd nlysis, nd mnuscript editing: Ds S Avilility of dt nd mterils The rw dt nd mterils re housed in the lortory of Dvis RL nd ville s pproprite. Finncil support nd sponsorship Oklhom Center for the Advncement of Science & Technology (HR14-007) (RLD); Oklhom Stte University Center for Helth Sciences, Intrmurl funds (RLD). Conflicts of interest All uthors declred tht there re no conflicts of interest. Ethicl pprovl nd consent to prticipte Not pplicle. Consent for puliction Not pplicle. Copyright The Author(s) REFERENCES 1. Wolf SA, Boddeke HW, Kettenmnn H. Microgli in Physiology nd Disese. Annu Rev Physiol 2017;79: Nimmerjhn A, Kirchhoff F, Helmchen F. Resting microglil cells re highly dynmic surveillnts of rin prenchym in vivo. Science 2005;308: Dvlos D, Grutzendler J, Yng G, Kim JV, Zuo Y, et l. ATP medites rpid microglil response to locl rin injury in vivo. Nt Neurosci 2005;8: Hong S, Dissing-Olesen L, Stevens B. New insights on the role of microgli in synptic pruning in helth nd disese. Curr Opin Neuroiol 2016;36: Werneurg S, Feinerg PA, Johnson KM, Schfer DP. A microgli-cytokine xis to modulte synptic connectivity nd function. Curr Opin Neuroiol 2017;47: Rogers JT, Morgnti JM, Bchstetter AD, Hudson CE, Peters MM, et l. CX3CR1 deficiency leds to impirment of hippocmpl cognitive function nd synptic plsticity. J Neurosci 2011;31: Schfer DP, Lehrmn EK, Stevens B. The qud-prtite synpse: microgli-synpse interctions in the developing nd mture CNS. Gli 2013;61: Slter MW, Stevens B. Microgli emerge s centrl plyers in rin disese. Nt Med 2017;23: Henek MT, Kummer MP, Ltz E. Innte immune ctivtion in neurodegenertive disese. Nt Rev Immunol 2014;14: Lee SC, Liu W, Dickson DW, Brosnn CF, Bermn JW. Cytokine production y humn fetl microgli nd strocytes. Differentil induction y lipopolyscchride nd IL-1 et. J Immunol 1993;150: Rustenhoven J, Prk TI, Schweder P, Scotter J, Correi J, et l. Isoltion of highly enriched primry humn microgli for functionl studies. Sci Rep 2016;6: Lokensgrd JR, Hu S, vn Fenem EM, Sheng WS, Peterson PK. Effect of thlidomide on chemokine production y humn microgli. J Infect Dis 2000;182: D Avers TG, Yu KO, Bermn JW. Expression of chemokines y humn fetl microgli fter tretment with the humn immunodeficiency virus type 1 protein Tt. J Neurovirol 2004;10: Liu J, Zho ML, Brosnn CF, Lee SC. Expression of type II nitric oxide synthse in primry humn strocytes nd microgli: role of IL- 1et nd IL-1 receptor ntgonist. J Immunol 1996;157: Prk J, Min JS, Kim B, Che UB, Yun JW, et l. Mitochondril ROS govern the LPS-induced pro-inflmmtory response in microgli cells y regulting MAPK nd NF-kppB pthwys. Neurosci Lett 2015;584: Cinciulli A, Drgone T, Clvello R, Porro C, Trott T, et l. IL-10 plys pivotl role in nti-inflmmtory effects of resvertrol in

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