{"paper_id":"1da0d98c-ca6b-4f97-8584-46d6c64e07be","body_text":"Increased Serum MMP-9 in Long-COVID May Reflect Activation of Microglia by SARS-CoV-2 Spike Protein | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Increased Serum MMP-9 in Long-COVID May Reflect Activation of Microglia by SARS-CoV-2 Spike Protein Duraisamy Kempuraj, Irene Tsilioni, Kristina K. Aenlle, Nancy G. Klimas, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4151696/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Long-COVID is a major health concern because many patients develop chronic neuropsychiatric symptoms, but the precise pathogenesis is unknown. Matrix metalloproteinase-9 (MMP-9) can disrupt neuronal connectivity and was elevated in patients with COVID-19. MMP-9 was measured in the serum of long COVID patients and healthy controls, as well as in the supernatant fluid of cultured human SV-40 microglia, by commercial ELISA. Results were analyzed with one-way ANOVA. MMP-9 in the serum of Long-COVID patients and supernatant fluid from cultured human microglia stimulated by recombinant SARS-CoV-2 Spike protein was assayed by ELISA. MMP-9 was significantly elevated in the serum of Long-COVID patients compared to healthy controls. Moreover, cultured human microglia released MMP-9 when stimulated by Spike protein. In conclusion, MMP-9 may contribute to the development of Long-COVID and serve both as a prognostic biomarker and as target for treatment. brain inflammation Long COVID mast cells microglia MMP-9 Figures Figure 1 Figure 2 Introduction Long COVID has been considered the “Next National Health Disaster” in the US (Phillips and Williams, 2021 ). As many as 50 per cent of those infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may develop Long-COVID (Thaweethai et al., 2023), especially Neuro-COVID (Ali et al., 2022 ; Almulla and Al-Hakeim, 2023 ) characterized cognitive dysfunction (Hadad et al., 2022 ). Long COVID may last up to two years.(Shanley et al., 2022 ) However, the precise pathogenesis of Long COVID has yet to be fully elucidated (Proal and VanElzakker, 2021 ). SARS-CoV-2 enters cells via its coronavirus spike protein binding to its cell surface receptor, angiotensin-converting enzyme 2 (ACE2) (Tai et al., 2020 ). SARS-CoV-2 Spike protein may enter the brain from the nose through the nasal neural mucosa following the olfactory nerve tract (Meinhardt et al., 2021 ). While the exact brain pathogenetic mechanisms remain unclear, evidence points to the involvement of neuroinflammation (Sodagar et al., 2022 ; Tremblay et al., 2022 ), especially perivascular inflammation (Lee et al., 2021 ) and blood-brain barrier (BBB) disruption (Bonetto et al., 2022 ; Lee et al., 2021 ), leading to neuronal damage (Zingaropoli et al., 2022 ). Autopsy studies of patients with COVID-19 showed severe neuronal loss in the capillaries of the choroid plexus (Yang et al., 2021 ), as well as neuronal necrosis and glial cell hyperplasia (Xu et al., 2024 ). A two-year longitudinal study using plasma proteomics to probe Long-COVID reported that pathways related to neuron generation and differentiation were persistently suppressed (Gu et al., 2023 ). A critical component of neuronal connectivity is the extracellular matrix (ECM) that can be disrupted by matrix metalloproteinases (MMPs). MMP-9 has emerged an important molecule in neuropsychiatric (Kaczmarek et al., 2023 ; Lopez-Navarro and Gutierrez, 2022 ) and neurodegenerative disorders (Beroun et al., 2019 ). MMP-9 can disrupt the polysaccharide scaffolding of the brain matrix and digest tight junction proteins, thus disrupting neuronal connectivity (Stawarski et al., 2014 ). MMP-9 can cause vascular inflammation and increase BBB permeability (Dhanda and Sandhir, 2018 ). MMP-9 levels were elevated in the serum of COVID-19 patients and were associated with disease severity (Ding et al., 2023 ; Savic et al., 2022 ). We investigated serum levels of MMP-9 in Long-COVID patients, and whether recombinant SARS-CoV-2 Spike protein could stimulate release of MMP-9 from cultured human microglia and mast cells. Methods Patients Patients (n = 15, 6 female and 9 male, mean age were 57 years old) were recruited from Southern Florida. Study participants were recruited from a companion longitudinal study of residents of South Florida who tested positive for SARS-CoV-2. Nova Southeastern University IRB No: 2020 − 590 ( Approved January 6, 2021; Expires January 11, 2025). Individuals were recruited from those who tested positive for COVID-19 in Broward County and were included in the Florida Department of Health (FDOH) Bureau of Epidemiology COVID-19 surveillance data, or in the records of Community Health of South Florida Inc. (CHI), a Federally Qualified Health Center in Miami-Dade County or in the records of participating community-based provider offices. The inclusion/exclusion criteria for unrecovered individuals were fatigue, as well as one additional symptom that began after positive SARS-Co-V-2 test and that the participant self-reported experiencing “a good bit of the time,” “most of the time,” or “all of the time”) during the past month. Individuals were 18–65-year-old and were able to consent to the phenotyping study. The Unrecovered group had moderate to severe illness as indicated by PROMIS 29 score of 45 or lower on the physical sub score, and fatigue that does not resolve with rest and one additional symptom from the CDC SI screener. Individuals were excluded from the study if they had medical or psychiatric conditions diagnosed prior to testing positive for SARS-CoV-2. Examples of exclusions included: severe chronic obstructive pulmonary disease, organ failure, chronic infection, rheumatic and chronic inflammatory disease, chronic lung disease, or major neurologic disease. In addition, the following were assessed during clinical visit and patients were excluded if there was evidence of abnormal diastolic function or cardiomyopathy and/or O 2 saturation of 92% or below on the 6-min exercise. Serum from healthy subjects (n = 20, 8 female and 12 male, mean age were 52 years old) was purchased from BioIvt Elevating Science (Hicksville, NY). Human Microglia Cell Culture The immortalized human microglia-SV40 cell line derived from primary human microglia was purchased from Applied Biological Materials Inc. (ABM Inc.; Richmond, BC, Canada) and cultured in Prigrow III medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in type I collagen-coated T25-flasks (BD PureCoat™ ECM Mimetic Cultureware Collagen I peptide plates, Becton Dickinson, Bedford, MA). Microglia-SV40 maintains their phenotype and proliferation rates for over 10 passages, during which all experiments were performed using multiple microglia thaws and sub-cultured cells. Experiments were carried out in type I collagen coated plates (BD). Cell viability was determined by trypan blue (0.4%) exclusion. Cell Treatments SV40 microglia (2.5 x 10 5 cells) were stimulated with recombinant full-length SARS-CoV-2 Spike protein (from Abcam, Waltham, MA, 10 ng/ml for 24 hr) and MMP-9 was measured in the supernatant fluid by ELISA (BioTechne, Minneapolis, MN) according to the manufacturer’s instructions. Control cells were treated with equal volume of culture medium in all experiments. Statistical Analysis All experimental conditions were performed in triplicate and all experiments were repeated at least three times (n = 3). Results from cultured cells are presented as mean ± SD. Comparisons between control and stimulated cells were performed using either parametric tests (unpaired 2-tailed, Student’s t -test, for independent samples) or Mann-Whitney non-parametric test depending on the normality of distribution that was checked with the Shapiro–Wilk’s test, followed by post-hoc analysis by Dunnett’s Multiple Comparison Test or the Wilcoxon post-hoc paired rank sum test. Comparisons among groups were performed with one-way analysis of Variance (ANOVA). All statistical analyses were performed by using GraphPad Prism 9.4.1. Results Here we show significantly increased (*p < 0.05, t-test) levels of MMP-9 in the serum of Long-COVID patients compared to healthy control subjects (Fig. 1 ). We also investigated whether SARS-CoV-2 Spike protein (1, 5, and10 ng/ml for 24 hr) could stimulate release of MMP-9 from SV-40 microglia. The Spike protein at 1, 5 and 10 ng/ml significantly (*p < 0.05) increased release of MMP-9 from microglia (Fig. 2 ). Neurotensin (NT) used as control significantly increased MMP-9 release compared to unstimulated control cells. Discussion Here we show that MMP-9 is elevated in the serum of Long-COVID patients. Elevated serum MMP-9 levels have been reported in COVID-19 and were associated with severity (Ding et al., 2023 ; Savic et al., 2022 ). MMP-9 polymorphisms were also reported to increase the susceptibility to COVID-19, especially when accompanied by neurologic symptoms (Bonetto et al., 2022 ). MMP-9 has also been associated with reduced BBB integrity (Bonetto et al., 2022 ; Rempe et al., 2016 ). We also show that microglia release MMP-9 when stimulated by SARS-CoV-2 Spike protein. We had previously reported that SARS-CoV-2 Spike protein stimulated cultured human microglia to secrete IL-1b, IL-18 and protein S100B, associated with brain damage (Tsilioni and Theoharides, 2023 ). Additional evidence indicates that the Spike protein can directly activate microglia (Jeong et al., 2022 ; Olajide et al., 2022 ; Samudyata et al., 2022 ) leading to proinflammatory effects. The neurological issues of Long-COVID may be attributed to the SARS-CoV-2 Spike protein (Theoharides and Kempuraj, 2023 ) since SARS-CoV-2 has not been shown to infect brain cells. In articular, a recent study demonstrated that SARS-CoV-2 has limited potential to proliferate in the brain and was unable to transmit between synaptic axons neurons in a human stem cell-derived neuronal culture system (Luczo et al., 2024 ). Perivascular inflammation with lymphocytic and microglial infiltration was noted in the brains of 52 deceased patients with COVID-19 (Wierzba-Bobrowicz et al., 2021 ). The duration of Long-COVID may depend on the length of antigen presence since it was reported that Spike protein was detected in CD16 + monocytes in Long- COVID patients up to 15–24 months post-infection(Patterson et al., 2021 ) and inside extracellular vesicles for up to one year (Craddock et al., 2023 ; Peluso et al., 2022 ). Recent papers reported that the SARS-CoV-2 Spike protein could be detected in Long-COVID patients for 6–12 months (Swank et al., 2023 ) and be present in “reservoirs”(Proal et al., 2023 ) including the brain. Microglia have been considered key players in the development of neuroinflammatory (Bachiller et al., 2018 ) and neurodegenerative disorders (Hickman et al., 2018 ; Perry et al., 2010 ). A recent meta-analysis of serum and plasma proteomic data indicated a significant association of COVID-19 with neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease (Mahin et al., 2024 ). There are limitations in this study since we do not know the original severity of COVID-19 in the patients studied. The source of MMP-9 is also not known. SARS-CoV-2 could stimulate release of MMP-9 from cultured macrophages (Murphy et al., 2023 ). Another paper reported that cultured human mast cells can also produce MMP-9 in response to an ionophore (Kimata et al., 2006 ). In conclusion, these results indicate that MMP-9 may possibly serve as a prognostic biomarker for development of Long-COVID and potential target for treatment. In fact, MMP-9 inhibitors have been considered for the treatment of traumatic brain injury (Sunny et al., 2024 ). In particular, the natural flavonoid nobiletin has been reported to inhibit MMP-9 (Kim et al., 2014 ). Declarations On behalf of all authors, the corresponding author states that there is no conflict of interest. All human studies have been approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments; and the specific national laws have been observed. Author Contribution KD performed the serum measurements. IT performed the in vitro studies and the supernatant fluid measurements and analyzed the results. KKA collected and stored the serum samples. NGK designed the clinical study, obtained HIRB approval and evaluated the patients. Acknowledgments This research was partly funded by a CDC grant 75D30120C09554 to NGK. “COVID 19 - Understanding the Post-Viral Phase (COVID-UPP)” and by donations from an anonymous family foundation to TCT. References Ali ST, Kang AK, Patel TR, Clark JR, Perez-Giraldo GS, Orban ZS, Lim PH, Jimenez M, Graham EL, Batra A, Liotta EM, Koralnik IJ (2022) Evolution of neurologic symptoms in non-hospitalized COVID-19 long haulers. Ann Clin Transl Neurol 9:950–961 Almulla AF, Al-Hakeim HK (2023) Editorial: Neuropsychiatric and neurodegenerative aspects of acute and long COVID. Front Mol Neurosci 16:1343930 Bachiller S, Jimenez-Ferrer I, Paulus A, Yang Y, Swanberg M, Deierborg T, Boza-Serrano A (2018) Microglia in Neurological Diseases: A Road Map to Brain-Disease Dependent-Inflammatory Response. Front Cell Neurosci 12:488 Beroun A, Mitra S, Michaluk P, Pijet B, Stefaniuk M, Kaczmarek L (2019) MMPs in learning and memory and neuropsychiatric disorders. Cell Mol Life Sci 76:3207–3228 Bonetto V, Pasetto L, Lisi I, Carbonara M, Zangari R, Ferrari E, Punzi V, Luotti S, Bottino N, Biagianti B, Moglia C, Fuda G, Gualtierotti R, Blasi F, Canetta C, Montano N, Tettamanti M, Camera G, Grimoldi M, Negro G, Rifino N, Calvo A, Brambilla P, Biroli F, Bandera A, Nobili A, Stocchetti N, Sessa M, Zanier ER (2022) Markers of blood-brain barrier disruption increase early and persistently in COVID-19 patients with neurological manifestations. Front Immunol 13:1070379 Craddock V, Mahajan A, Spikes L, Krishnamachary B, Ram AK, Kumar A, Chen L, Chalise P, Dhillon NK (2023) Persistent circulation of soluble and extracellular vesicle-linked Spike protein in individuals with postacute sequelae of COVID-19. J Med Virol 95:e28568 Dhanda S, Sandhir R (2018) Blood-Brain Barrier Permeability Is Exacerbated in Experimental Model of Hepatic Encephalopathy via MMP-9 Activation and Downregulation of Tight Junction Proteins. Mol Neurobiol 55:3642–3659 Ding L, Guo H, Zhang C, Jin H, Guo X, Li T (2023) Elevated matrix metalloproteinase–9 expression is associated with COVID–19 severity: A meta–analysis. Exp Ther Med 26:545 Gu X, Wang S, Zhang W, Li C, Guo L, Wang Z, Li H, Zhang H, Zhou Y, Liang W, Li H, Liu Y, Wang Y, Huang L, Dong T, Zhang D, Wong CCL, Cao B (2023) Probing long COVID through a proteomic lens: a comprehensive two-year longitudinal cohort study of hospitalised survivors. EBioMedicine 98:104851 Hadad R, Khoury J, Stanger C, Fisher T, Schneer S, Ben-Hayun R, Possin K, Valcour V, Aharon-Peretz J, Adir Y (2022) Cognitive dysfunction following COVID-19 infection. J Neurovirol 28:430–437 Hickman S, Izzy S, Sen P, Morsett L, El KJ (2018) Microglia in neurodegeneration. Nat Neurosci 21:1359–1369 Jeong GU, Lyu J, Kim KD, Chung YC, Yoon GY, Lee S, Hwang I, Shin WH, Ko J, Lee JY, Kwon YC (2022) SARS-CoV-2 Infection of Microglia Elicits Proinflammatory Activation and Apoptotic Cell Death. Microbiol Spectr 10:e0109122 Kaczmarek KT, Protokowicz K, Kaczmarek L (2023) Matrix metalloproteinase-9: A magic drug target in neuropsychiatry? J Neurochem Kim JJ, Korm S, Kim WS, Kim OS, Lee JS, Min HG, Chin YW, Cha HJ (2014) Nobiletin suppresses MMP-9 expression through modulation of p38 MAPK activity in human dermal fibrobalsts. Biol Pharm Bull 37:158–163 Kimata M, Ishizaki M, Tanaka H, Nagai H, Inagaki N (2006) Production of matrix metalloproteinases in human cultured mast cells: involvement of protein kinase C-mitogen activated protein kinase kinase-extracellular signal-regulated kinase pathway. Allergol Int 55:67–76 Lee MH, Perl DP, Nair G, Li W, Maric D, Murray H, Dodd SJ, Koretsky AP, Watts JA, Cheung V, Masliah E, Horkayne-Szakaly I, Jones R, Stram MN, Moncur J, Hefti M, Folkerth RD, Nath A (2021) Microvascular Injury in the Brains of Patients with Covid-19. N Engl J Med 384:481–483 Lopez-Navarro ER, Gutierrez J (2022) Metalloproteinases and their inhibitors in neurological disease. Naunyn Schmiedebergs Arch Pharmacol 395:27–38 Luczo JM, Edwards SJ, Ardipradja K, Suen WW, Au GG, Marsh GA, Godde N, Rootes CL, Bingham J, Sundaramoorthy V (2024) SARS-CoV and SARS-CoV-2 display limited neuronal infection and lack the ability to transmit within synaptically connected axons in stem cell-derived human neurons. J Neurovirol Mahin A, Soman SP, Modi PK, Raju R, Keshava Prasad TS, Abhinand CS (2024) Meta-analysis of the serum/plasma proteome identifies significant associations between COVID-19 with Alzheimer's/Parkinson's diseases. J Neurovirol Meinhardt J, Radke J, Dittmayer C, Franz J, Thomas C, Mothes R, Laue M, Schneider J, Brunink S, Greuel S, Lehmann M, Hassan O, Aschman T, Schumann E, Chua RL, Conrad C, Eils R, Stenzel W, Windgassen M, Rossler L, Goebel HH, Gelderblom HR, Martin H, Nitsche A, Schulz-Schaeffer WJ, Hakroush S, Winkler MS, Tampe B, Scheibe F, Kortvelyessy P, Reinhold D, Siegmund B, Kuhl AA, Elezkurtaj S, Horst D, Oesterhelweg L, Tsokos M, Ingold-Heppner B, Stadelmann C, Drosten C, Corman VM, Radbruch H (2021) and F.L. Heppner. Olfactory transmucosal SARS-CoV-2 invasion as a port of central nervous system entry in individuals with COVID-19. Nat Neurosci 24:168–175 Murphy SL, Halvorsen B, Holter JC, Huse C, Tveita A, Troseid M, Hoel H, Kildal AB, Holten AR, Lerum TV, Skjonsberg OH, Michelsen AE, Aalokken TM, T.N., Nor-Solidarity Consortium K, Tonby A, Lind S, Dudman BK, Granerud L, Heggelund S, Boe AM, Dyrholt-Riise P, Aukrust A, Barratt-Due T, Ueland, Dahl TB (2023) Circulating markers of extracellular matrix remodelling in severe COVID-19 patients. J Intern Med 294:784–797 Olajide OA, Iwuanyanwu VU, Adegbola OD, Al-Hindawi AA (2022) SARS-CoV-2 Spike Glycoprotein S1 Induces Neuroinflammation in BV-2 Microglia. Mol Neurobiol 59:445–458 Patterson BK, Francisco EB, Yogendra R, Long E, Pise A, Rodrigues H, Hall E, Herrera M, Parikh P, Guevara-Coto J, Triche TJ, Scott P, Hekmati S, Maglinte D, Chang X, Mora-Rodriguez RA, Mora J (2021) Persistence of SARS CoV-2 S1 Protein in CD16 + Monocytes in Post-Acute Sequelae of COVID-19 (PASC) up to 15 Months Post-Infection. Front Immunol 12:746021 Peluso MJ, Deeks SG, Mustapic M, Kapogiannis D, Henrich TJ, Lu S, Goldberg SA, Hoh R, Chen JY, Martinez EO, Kelly JD, Martin JN, Goetzl EJ (2022) SARS-CoV-2 and Mitochondrial Proteins in Neural-Derived Exosomes of COVID-19. Ann Neurol 91:772–781 Perry VH, Nicoll JA, Holmes C (2010) Microglia in neurodegenerative disease. Nat Rev Neurol 6:193–201 Phillips S, Williams MA (2021) Confronting Our Next National Health Disaster - Long-Haul Covid. N Engl J Med 385:577–579 Proal AD, VanElzakker MB (2021) Long COVID or Post-acute Sequelae of COVID-19 (PASC): An Overview of Biological Factors That May Contribute to Persistent Symptoms. Front Microbiol 12:698169 Proal AD, VanElzakker MB, Aleman S, Bach K, Boribong BP, Buggert M, Cherry S, Chertow DS, Davies HE, Dupont CL, Deeks SG, Eimer W, Ely EW, Fasano A, Freire M, Geng LN, Griffin DE, Henrich TJ, Iwasaki A, Izquierdo-Garcia D, Locci M, Mehandru S, Painter MM, Peluso MJ, Pretorius E, Price DA, Putrino D, Scheuermann RH, Tan GS, Tanzi RE, VanBrocklin HF, Yonker LM, Wherry EJ (2023) Author Correction: SARS-CoV-2 reservoir in post-acute sequelae of COVID-19 (PASC). Nat Immunol 24:1778 Rempe RG, Hartz AM, Bauer B (2016) Matrix metalloproteinases in the brain and blood-brain barrier: versatile breakers and makers. J Cereb Blood Flow Metab 36:1481–14507 Samudyata AO, Oliveira S, Malwade N, Rufino de Sousa SK, Goparaju J, Gracias F, Orhan L, Steponaviciute M, Schalling SD, Sheridan RH, Perlis AG, Rothfuchs, Sellgren CM (2022) SARS-CoV-2 promotes microglial synapse elimination in human brain organoids. Mol Psychiatry 27:3939–3950 Savic G, Stevanovic I, Mihajlovic D, Jurisevic M, Gajovic N, Jovanovic I, Ninkovic M (2022) MMP-9/BDNF ratio predicts more severe COVID-19 outcomes. Int J Med Sci 19:1903–1911 Shanley JE, Valenciano AF, Timmons G, Miner AE, Kakarla V, Rempe T, Yang JH, Gooding A, Norman MA, Banks SJ, Ritter ML, Ellis RJ, Horton L, Graves JS (2022) Longitudinal evaluation of neurologic-post acute sequelae SARS-CoV-2 infection symptoms. Ann Clin Transl Neurol 9:995–1010 Sodagar A, Javed R, Tahir H, Razak SIA, Shakir M, Naeem M, Yusof AHA, Sagadevan S, Hazafa A, Uddin J, Khan A, Al-Harrasi A (2022) Pathological Features and Neuroinflammatory Mechanisms of SARS-CoV-2 in the Brain and Potential Therapeutic Approaches. Biomolecules 12 Stawarski M, Stefaniuk M, Wlodarczyk J (2014) Matrix metalloproteinase-9 involvement in the structural plasticity of dendritic spines. Front Neuroanat 8:68 Sunny A, James RR, Menon SR, Rayaroth S, Daniel A, Thompson NA, Tharakan B (2024) Matrix Metalloproteinase-9 inhibitors as therapeutic drugs for traumatic brain injury. Neurochem Int 172:105642 Swank Z, Senussi Y, Manickas-Hill Z, Yu XG, Li JZ, Alter G, Walt DR (2023) Persistent Circulating Severe Acute Respiratory Syndrome Coronavirus 2 Spike Is Associated With Post-acute Coronavirus Disease 2019 Sequelae. Clin Infect Dis 76:e487–e490 Tai W, He L, Zhang X, Pu J, Voronin D, Jiang S, Zhou Y, Du L (2020) Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol 17:613–620 Thaweethai, T., S.E. Jolley, E.W. Karlson, E.B. Levitan, B. Levy, G.A. McComsey, L.McCorkell, G.N. Nadkarni, S. Parthasarathy, U. Singh, T.A. Walker, C.A. Selvaggi,D.J. Shinnick, C.C.M. Schulte, R. Atchley-Challenner, G.A. Alba, R. Alicic, N. Altman,K. Anglin, U. Argueta, H. Ashktorab, G. Baslet, I.V. Bassett, L. Bateman, B. Bedi,S. Bhattacharyya, M.A. Bind, A.L. Blomkalns, H. Bonilla, P.A. Bush, M. Castro, J.Chan, A.W. Charney, P. Chen, L.B. Chibnik, H.Y. Chu, R.G. Clifton, M.M. Costantine,S.K. Cribbs, S.I. Davila Nieves, S.G. Deeks, A. Duven, I.F. Emery, N. Erdmann, K.M.Erlandson, K.C. Ernst, R. Farah-Abraham, C.E. Farner, E.M. Feuerriegel, J. Fleurimont,V. Fonseca, N. Franko, V. Gainer, J.C. Gander, E.M. Gardner, L.N. Geng, K.S. Gibson,M. Go, J.D. Goldman, H. Grebe, F.L. Greenway, M. Habli, J. Hafner, J.E. Han, K.A.Hanson, J. Heath, C. Hernandez, R. Hess, S.L. Hodder, M.K. Hoffman, S.E. Hoover, B.Huang, B.L. Hughes, P. Jagannathan, J. John, M.R. Jordan, S.D. Katz, E.S. Kaufman,J.D. Kelly, S.W. Kelly, M.M. Kemp, J.P. Kirwan, J.D. Klein, K.S. Knox, J.A. Krishnan,A. Kumar, A.O. Laiyemo, A.A. Lambert, M. Lanca, J.K. Lee-Iannotti, B.P. Logarbo, M.T.Longo, C.A. Luciano, K. Lutrick, J.H. Maley, J.G. Marathe, V. Marconi, G.D. Marshall,C.F. Martin, Y. Matusov, A. Mehari, H. Mendez-Figueroa, R. Mermelstein, T.D. Metz,R. Morse, J. Mosier, C. Mouchati, J. Mullington, S.N. Murphy, R.B. Neuman, J.Z. Nikolich,I. Ofotokun, E. Ojemakinde, A. Palatnik, K. Palomares, T. Parimon, S. Parry, J.E.Patterson, T.F. Patterson, R.E. Patzer, M.J. Peluso, P. Pemu, C.M. Pettker, B.A. Plunkett,K. Pogreba-Brown, A. Poppas, J.G. Quigley, U. Reddy, R. Reece, H. Reeder, W.B. Reeves,E.M. Reiman, F. Rischard, J. Rosand, D.J. Rouse, A. Ruff, G. Saade, G.J. Sandoval,S.M. Schlater, F. Shepherd, Z.A. Sherif, H. Simhan, N.G. Singer, D.W. Skupski, A.Sowles, J.A. Sparks, F.I. Sukhera, B.S. Taylor, L. Teunis, R.J. Thomas, J.M. Thorp,P. Thuluvath, A. Ticotsky, A.T. Tita, K.R. Tuttle, A.E. Urdaneta, D. Valdivieso, T.M.VanWagoner, A. Vasey, M. Verduzco-Gutierrez, Z.S. Wallace, H.D. Ward, D.E. Warren,S.J. Weiner, S. Welch, S.W. Whiteheart, Z. Wiley, J.P. Wisnivesky, L.M. Yee, S. Zisis,L.I. Horwitz, A.S. Foulkes, and R. Consortium. 2023. Development of a Definition of Postacute Sequelae of SARS-CoV-2 Infection. JAMA 329:1934–1946 Theoharides TC, Kempuraj D (2023) Role of SARS-CoV-2 Spike-Protein-Induced Activation of Microglia and Mast Cells in the Pathogenesis of Neuro-COVID. Cells 12:688 Tremblay ME, Madore C, Tian L, Verkhratsky A (2022) Editorial: Role of Neuroinflammation in the Neuropsychiatric and Neurological Aspects of COVID-19. Front Cell Neurosci 16:840121 Tsilioni I, Theoharides TC (2023) Recombinant SARS-CoV-2 Spike Protein and Its Receptor Binding Domain Stimulate Release of Different Pro-Inflammatory Mediators via Activation of Distinct Receptors on Human Microglia Cells. Mol Neurobiol Wierzba-Bobrowicz T, Krajewski P, Tarka S, Acewicz A, Felczak P, Stepien T, M PG, Grzegorczyk M (2021) Neuropathological analysis of the brains of fifty-two patients with COVID-19. Folia Neuropathol 59:219–231 Xu Z, Wang H, Jiang S, Teng J, Zhou D, Chen Z, Wen C, Xu Z (2024) Brain Pathology in COVID-19: Clinical Manifestations and Potential Mechanisms. Neurosci Bull 40:383–400 Yang AC, Kern F, Losada PM, Agam MR, Maat CA, Schmartz GP, Fehlmann T, Stein JA, Schaum N, Lee DP, Calcuttawala K, Vest RT, Berdnik D, Lu N, Hahn O, Gate D, McNerney MW, Channappa D, Cobos I, Ludwig N, Schulz-Schaeffer WJ, Keller A, Wyss-Coray T (2021) Dysregulation of brain and choroid plexus cell types in severe COVID-19. Nature 595:565–571 Zingaropoli MA, Iannetta M, Piermatteo L, Pasculli P, Latronico T, Mazzuti L, Campogiani L, Duca L, Ferraguti G, De Michele M, Galardo G, Pugliese F, Antonelli G, Andreoni M, Sarmati L, Lichtner M, Turriziani O, Ceccherini-Silberstein F, Liuzzi GM, Mastroianni CM, Ciardi MR (2022) Neuro-Axonal Damage and Alteration of Blood-Brain Barrier Integrity in COVID-19 Patients. Cells 11:2480 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-4151696\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Short Report\",\"associatedPublications\":[],\"authors\":[{\"id\":285147278,\"identity\":\"b3e86c2c-222c-45ad-b2c5-bfb97db95491\",\"order_by\":0,\"name\":\"Duraisamy Kempuraj\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Duraisamy\",\"middleName\":\"\",\"lastName\":\"Kempuraj\",\"suffix\":\"\"},{\"id\":285147279,\"identity\":\"7b897e62-a7fd-476f-aec8-c74779ca64a9\",\"order_by\":1,\"name\":\"Irene Tsilioni\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Tufts University School of Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Irene\",\"middleName\":\"\",\"lastName\":\"Tsilioni\",\"suffix\":\"\"},{\"id\":285147280,\"identity\":\"53f3e05d-d0f9-4ea4-9a1f-69c4aa2f840b\",\"order_by\":2,\"name\":\"Kristina K. Aenlle\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Kristina\",\"middleName\":\"K.\",\"lastName\":\"Aenlle\",\"suffix\":\"\"},{\"id\":285147281,\"identity\":\"b4baeaa4-95a5-4611-adff-d647af1fc673\",\"order_by\":3,\"name\":\"Nancy G. Klimas\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Nancy\",\"middleName\":\"G.\",\"lastName\":\"Klimas\",\"suffix\":\"\"},{\"id\":285147282,\"identity\":\"d874c47e-c6ca-4541-a320-15c6a8b65984\",\"order_by\":4,\"name\":\"Theoharis C. Theoharides\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYFACHgYGxgYGBn4oF8QmoIENqkWygWQtBgeI1WIv33vwwccddvnGt5uPSfxgsJHdcICgLXzJhjPPJFtuu3MsTbKHIc2YCC08ZtK8bcwGZjdyjA14GA4nEqPF/DdvW72B8YwcY8M/DP+J0mLGzNt22MBAIsfwMQ/DASK0HMsxlpzZdtxA4kZa4mMZg2TjmYS0sDefMfzwsa3agH9G8oGDbyrsZPsIaUEDBqQpHwWjYBSMglGAAwAAT/I93Szw1pgAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Theoharis\",\"middleName\":\"C.\",\"lastName\":\"Theoharides\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-03-22 19:31:41\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4151696/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4151696/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":53877101,\"identity\":\"a8bbb7ab-5ad7-4579-b8d9-7609dff79f6d\",\"added_by\":\"auto\",\"created_at\":\"2024-04-01 16:43:20\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":90784,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSerum levels of MMP-9. Scattergram of serum values of MMP-9. MMP-9 was measured in the serum of Long-COVID (n=15, mean age 55 years) and healthy control subjects (n=20, mean age 52 years) using ELISA kit. *p\\u0026lt;0.05; t-test compared to control subjects.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4151696/v1/91383eeb6c4798b2401e6675.jpg\"},{\"id\":53875561,\"identity\":\"006206cf-1fc4-4c32-9b50-a1fe71003ffe\",\"added_by\":\"auto\",\"created_at\":\"2024-04-01 16:35:20\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":38180,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eRecombinant SARS-CoV-2 Spike protein stimulates release of MMP-9 from human microglia. SV-40 microglia (2.5 x 10\\u003csup\\u003e5\\u003c/sup\\u003e cells) were stimulated with recombinant full-length SARS-CoV-2 Spike protein (1, 5, 10 ng/ml for 24 hr) and MMP-9 was measured in the supernatant fluid by ELISA. NT used as control significantly increased MMP-9 release compared to unstimulated control cells. C=control. All conditions were performed in triplicate for each dataset and repeated 3 times (n=3). Results are presented as mean ± standard error of the mean (SEM). One-Way ANOVA showed significant difference among means (p=0,0007) listed here as p\\u0026lt;0.05. \\u0026nbsp;\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4151696/v1/73f89845eb993787d56eb900.jpg\"},{\"id\":54934554,\"identity\":\"f9007d42-8438-4f7e-8bfb-5037bafa61f8\",\"added_by\":\"auto\",\"created_at\":\"2024-04-18 20:10:32\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":264827,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4151696/v1/37e241d7-d4af-48eb-a2c8-26ba02e02bee.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Increased Serum MMP-9 in Long-COVID May Reflect Activation of Microglia by SARS-CoV-2 Spike Protein\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eLong COVID has been considered the \\u0026ldquo;Next National Health Disaster\\u0026rdquo; in the US (Phillips and Williams, \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). As many as 50 per cent of those infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may develop Long-COVID (Thaweethai et al., 2023), especially Neuro-COVID (Ali et al., \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Almulla and Al-Hakeim, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e) characterized cognitive dysfunction (Hadad et al., \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Long COVID may last up to two years.(Shanley et al., \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e) However, the precise pathogenesis of Long COVID has yet to be fully elucidated (Proal and VanElzakker, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). SARS-CoV-2 enters cells via its coronavirus spike protein binding to its cell surface receptor, angiotensin-converting enzyme 2 (ACE2) (Tai et al., \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). SARS-CoV-2 Spike protein may enter the brain from the nose through the nasal neural mucosa following the olfactory nerve tract (Meinhardt et al., \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). While the exact brain pathogenetic mechanisms remain unclear, evidence points to the involvement of neuroinflammation (Sodagar et al., \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Tremblay et al., \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e), especially perivascular inflammation (Lee et al., \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e) and blood-brain barrier (BBB) disruption (Bonetto et al., \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Lee et al., \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e), leading to neuronal damage (Zingaropoli et al., \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eAutopsy studies of patients with COVID-19 showed severe neuronal loss in the capillaries of the choroid plexus (Yang et al., \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e), as well as neuronal necrosis and glial cell hyperplasia (Xu et al., \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). A two-year longitudinal study using plasma proteomics to probe Long-COVID reported that pathways related to neuron generation and differentiation were persistently suppressed (Gu et al., \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eA critical component of neuronal connectivity is the extracellular matrix (ECM) that can be disrupted by matrix metalloproteinases (MMPs). MMP-9 has emerged an important molecule in neuropsychiatric (Kaczmarek et al., \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Lopez-Navarro and Gutierrez, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e) and neurodegenerative disorders (Beroun et al., \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). MMP-9 can disrupt the polysaccharide scaffolding of the brain matrix and digest tight junction proteins, thus disrupting neuronal connectivity (Stawarski et al., \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e). MMP-9 can cause vascular inflammation and increase BBB permeability (Dhanda and Sandhir, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). MMP-9 levels were elevated in the serum of COVID-19 patients and were associated with disease severity (Ding et al., \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Savic et al., \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eWe investigated serum levels of MMP-9 in Long-COVID patients, and whether recombinant SARS-CoV-2 Spike protein could stimulate release of MMP-9 from cultured human microglia and mast cells.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePatients\\u003c/h2\\u003e \\u003cp\\u003ePatients (n\\u0026thinsp;=\\u0026thinsp;15, 6 female and 9 male, mean age were 57 years old) were recruited from Southern Florida. Study participants were recruited from a companion longitudinal study of residents of South Florida who tested positive for SARS-CoV-2. Nova Southeastern University IRB No: 2020\\u0026thinsp;\\u0026minus;\\u0026thinsp;590 \\u003cb\\u003e(\\u003c/b\\u003eApproved January 6, 2021; Expires January 11, 2025). Individuals were recruited from those who tested positive for COVID-19 in Broward County and were included in the Florida Department of Health (FDOH) Bureau of Epidemiology COVID-19 surveillance data, or in the records of Community Health of South Florida Inc. (CHI), a Federally Qualified Health Center in Miami-Dade County or in the records of participating community-based provider offices. The inclusion/exclusion criteria for unrecovered individuals were fatigue, as well as one additional symptom that began after positive SARS-Co-V-2 test and that the participant self-reported experiencing \\u0026ldquo;a good bit of the time,\\u0026rdquo; \\u0026ldquo;most of the time,\\u0026rdquo; or \\u0026ldquo;all of the time\\u0026rdquo;) during the past month. Individuals were 18\\u0026ndash;65-year-old and were able to consent to the phenotyping study. The Unrecovered group had moderate to severe illness as indicated by PROMIS 29 score of 45 or lower on the physical sub score, and fatigue that does not resolve with rest and one additional symptom from the CDC SI screener. Individuals were excluded from the study if they had medical or psychiatric conditions diagnosed prior to testing positive for SARS-CoV-2. Examples of exclusions included: severe chronic obstructive pulmonary disease, organ failure, chronic infection, rheumatic and chronic inflammatory disease, chronic lung disease, or major neurologic disease. In addition, the following were assessed during clinical visit and patients were excluded if there was evidence of abnormal diastolic function or cardiomyopathy and/or O\\u003csub\\u003e2\\u003c/sub\\u003e saturation of 92% or below on the 6-min exercise. Serum from healthy subjects (n\\u0026thinsp;=\\u0026thinsp;20, 8 female and 12 male, mean age were 52 years old) was purchased from BioIvt Elevating Science (Hicksville, NY).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eHuman Microglia Cell Culture\\u003c/h2\\u003e \\u003cp\\u003eThe immortalized human microglia-SV40 cell line derived from primary human microglia was purchased from Applied Biological Materials Inc. (ABM Inc.; Richmond, BC, Canada) and cultured in Prigrow III medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in type I collagen-coated T25-flasks (BD PureCoat\\u0026trade; ECM Mimetic Cultureware Collagen I peptide plates, Becton Dickinson, Bedford, MA). Microglia-SV40 maintains their phenotype and proliferation rates for over 10 passages, during which all experiments were performed using multiple microglia thaws and sub-cultured cells. Experiments were carried out in type I collagen coated plates (BD). Cell viability was determined by trypan blue (0.4%) exclusion.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCell Treatments\\u003c/h2\\u003e \\u003cp\\u003eSV40 microglia (2.5 x 10\\u003csup\\u003e5\\u003c/sup\\u003e cells) were stimulated with recombinant full-length SARS-CoV-2 Spike protein (from Abcam, Waltham, MA, 10 ng/ml for 24 hr) and MMP-9 was measured in the supernatant fluid by ELISA (BioTechne, Minneapolis, MN) according to the manufacturer\\u0026rsquo;s instructions. Control cells were treated with equal volume of culture medium in all experiments.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical Analysis\\u003c/h2\\u003e \\u003cp\\u003eAll experimental conditions were performed in triplicate and all experiments were repeated at least three times (n\\u0026thinsp;=\\u0026thinsp;3). Results from cultured cells are presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD. Comparisons between control and stimulated cells were performed using either parametric tests (unpaired 2-tailed, Student\\u0026rsquo;s \\u003cem\\u003et\\u003c/em\\u003e-test, for independent samples) or Mann-Whitney non-parametric test depending on the normality of distribution that was checked with the Shapiro\\u0026ndash;Wilk\\u0026rsquo;s test, followed by post-hoc analysis by Dunnett\\u0026rsquo;s Multiple Comparison Test or the Wilcoxon post-hoc paired rank sum test. Comparisons among groups were performed with one-way analysis of Variance (ANOVA). All statistical analyses were performed by using GraphPad Prism 9.4.1.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003eHere we show significantly increased (*p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, t-test) levels of MMP-9 in the serum of Long-COVID patients compared to healthy control subjects (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eWe also investigated whether SARS-CoV-2 Spike protein (1, 5, and10 ng/ml for 24 hr) could stimulate release of MMP-9 from SV-40 microglia. The Spike protein at 1, 5 and 10 ng/ml significantly (*p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) increased release of MMP-9 from microglia (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Neurotensin (NT) used as control significantly increased MMP-9 release compared to unstimulated control cells.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003eHere we show that MMP-9 is elevated in the serum of Long-COVID patients. Elevated serum MMP-9 levels have been reported in COVID-19 and were associated with severity (Ding et al., \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Savic et al., \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). MMP-9 polymorphisms were also reported to increase the susceptibility to COVID-19, especially when accompanied by neurologic symptoms (Bonetto et al., \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). MMP-9 has also been associated with reduced BBB integrity (Bonetto et al., \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Rempe et al., \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). We also show that microglia release MMP-9 when stimulated by SARS-CoV-2 Spike protein. We had previously reported that SARS-CoV-2 Spike protein stimulated cultured human microglia to secrete IL-1b, IL-18 and protein S100B, associated with brain damage (Tsilioni and Theoharides, \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Additional evidence indicates that the Spike protein can directly activate microglia (Jeong et al., \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Olajide et al., \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Samudyata et al., \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e) leading to proinflammatory effects.\\u003c/p\\u003e \\u003cp\\u003eThe neurological issues of Long-COVID may be attributed to the SARS-CoV-2 Spike protein (Theoharides and Kempuraj, \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e) since SARS-CoV-2 has not been shown to infect brain cells. In articular, a recent study demonstrated that SARS-CoV-2 has limited potential to proliferate in the brain and was unable to transmit between synaptic axons neurons in a human stem cell-derived neuronal culture system (Luczo et al., \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Perivascular inflammation with lymphocytic and microglial infiltration was noted in the brains of 52 deceased patients with COVID-19 (Wierzba-Bobrowicz et al., \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). The duration of Long-COVID may depend on the length of antigen presence since it was reported that Spike protein was detected in CD16\\u003csup\\u003e+\\u003c/sup\\u003e monocytes in Long- COVID patients up to 15\\u0026ndash;24 months post-infection(Patterson et al., \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e) and inside extracellular vesicles for up to one year (Craddock et al., \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Peluso et al., \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Recent papers reported that the SARS-CoV-2 Spike protein could be detected in Long-COVID patients for 6\\u0026ndash;12 months (Swank et al., \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e) and be present in \\u0026ldquo;reservoirs\\u0026rdquo;(Proal et al., \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e) including the brain. Microglia have been considered key players in the development of neuroinflammatory (Bachiller et al., \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e) and neurodegenerative disorders (Hickman et al., \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Perry et al., \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e). A recent meta-analysis of serum and plasma proteomic data indicated a significant association of COVID-19 with neurodegenerative diseases such as Alzheimer\\u0026rsquo;s disease and Parkinson\\u0026rsquo;s disease (Mahin et al., \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThere are limitations in this study since we do not know the original severity of COVID-19 in the patients studied. The source of MMP-9 is also not known. SARS-CoV-2 could stimulate release of MMP-9 from cultured macrophages (Murphy et al., \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Another paper reported that cultured human mast cells can also produce MMP-9 in response to an ionophore (Kimata et al., \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eIn conclusion, these results indicate that MMP-9 may possibly serve as a prognostic biomarker for development of Long-COVID and potential target for treatment. In fact, MMP-9 inhibitors have been considered for the treatment of traumatic brain injury (Sunny et al., \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). In particular, the natural flavonoid nobiletin has been reported to inhibit MMP-9 (Kim et al., \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003eOn behalf of all authors, the corresponding author states that there is no conflict of interest. All human studies have been approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments; and the specific national laws have been observed.\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eKD performed the serum measurements. IT performed the in vitro studies and the supernatant fluid measurements and analyzed the results. KKA collected and stored the serum samples. NGK designed the clinical study, obtained HIRB approval and evaluated the patients.\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgments\\u003c/h2\\u003e \\u003cp\\u003eThis research was partly funded by a CDC grant 75D30120C09554 to NGK. \\u0026ldquo;COVID 19 - Understanding the Post-Viral Phase (COVID-UPP)\\u0026rdquo; and by donations from an anonymous family foundation to TCT.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAli ST, Kang AK, Patel TR, Clark JR, Perez-Giraldo GS, Orban ZS, Lim PH, Jimenez M, Graham EL, Batra A, Liotta EM, Koralnik IJ (2022) Evolution of neurologic symptoms in non-hospitalized COVID-19 long haulers. Ann Clin Transl Neurol 9:950\\u0026ndash;961\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAlmulla AF, Al-Hakeim HK (2023) Editorial: Neuropsychiatric and neurodegenerative aspects of acute and long COVID. Front Mol Neurosci 16:1343930\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBachiller S, Jimenez-Ferrer I, Paulus A, Yang Y, Swanberg M, Deierborg T, Boza-Serrano A (2018) Microglia in Neurological Diseases: A Road Map to Brain-Disease Dependent-Inflammatory Response. Front Cell Neurosci 12:488\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBeroun A, Mitra S, Michaluk P, Pijet B, Stefaniuk M, Kaczmarek L (2019) MMPs in learning and memory and neuropsychiatric disorders. Cell Mol Life Sci 76:3207\\u0026ndash;3228\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBonetto V, Pasetto L, Lisi I, Carbonara M, Zangari R, Ferrari E, Punzi V, Luotti S, Bottino N, Biagianti B, Moglia C, Fuda G, Gualtierotti R, Blasi F, Canetta C, Montano N, Tettamanti M, Camera G, Grimoldi M, Negro G, Rifino N, Calvo A, Brambilla P, Biroli F, Bandera A, Nobili A, Stocchetti N, Sessa M, Zanier ER (2022) Markers of blood-brain barrier disruption increase early and persistently in COVID-19 patients with neurological manifestations. Front Immunol 13:1070379\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCraddock V, Mahajan A, Spikes L, Krishnamachary B, Ram AK, Kumar A, Chen L, Chalise P, Dhillon NK (2023) Persistent circulation of soluble and extracellular vesicle-linked Spike protein in individuals with postacute sequelae of COVID-19. J Med Virol 95:e28568\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDhanda S, Sandhir R (2018) Blood-Brain Barrier Permeability Is Exacerbated in Experimental Model of Hepatic Encephalopathy via MMP-9 Activation and Downregulation of Tight Junction Proteins. Mol Neurobiol 55:3642\\u0026ndash;3659\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDing L, Guo H, Zhang C, Jin H, Guo X, Li T (2023) Elevated matrix metalloproteinase\\u0026ndash;9 expression is associated with COVID\\u0026ndash;19 severity: A meta\\u0026ndash;analysis. Exp Ther Med 26:545\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGu X, Wang S, Zhang W, Li C, Guo L, Wang Z, Li H, Zhang H, Zhou Y, Liang W, Li H, Liu Y, Wang Y, Huang L, Dong T, Zhang D, Wong CCL, Cao B (2023) Probing long COVID through a proteomic lens: a comprehensive two-year longitudinal cohort study of hospitalised survivors. EBioMedicine 98:104851\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHadad R, Khoury J, Stanger C, Fisher T, Schneer S, Ben-Hayun R, Possin K, Valcour V, Aharon-Peretz J, Adir Y (2022) Cognitive dysfunction following COVID-19 infection. J Neurovirol 28:430\\u0026ndash;437\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHickman S, Izzy S, Sen P, Morsett L, El KJ (2018) Microglia in neurodegeneration. Nat Neurosci 21:1359\\u0026ndash;1369\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJeong GU, Lyu J, Kim KD, Chung YC, Yoon GY, Lee S, Hwang I, Shin WH, Ko J, Lee JY, Kwon YC (2022) SARS-CoV-2 Infection of Microglia Elicits Proinflammatory Activation and Apoptotic Cell Death. Microbiol Spectr 10:e0109122\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKaczmarek KT, Protokowicz K, Kaczmarek L (2023) Matrix metalloproteinase-9: A magic drug target in neuropsychiatry? J Neurochem\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKim JJ, Korm S, Kim WS, Kim OS, Lee JS, Min HG, Chin YW, Cha HJ (2014) Nobiletin suppresses MMP-9 expression through modulation of p38 MAPK activity in human dermal fibrobalsts. Biol Pharm Bull 37:158\\u0026ndash;163\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKimata M, Ishizaki M, Tanaka H, Nagai H, Inagaki N (2006) Production of matrix metalloproteinases in human cultured mast cells: involvement of protein kinase C-mitogen activated protein kinase kinase-extracellular signal-regulated kinase pathway. Allergol Int 55:67\\u0026ndash;76\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLee MH, Perl DP, Nair G, Li W, Maric D, Murray H, Dodd SJ, Koretsky AP, Watts JA, Cheung V, Masliah E, Horkayne-Szakaly I, Jones R, Stram MN, Moncur J, Hefti M, Folkerth RD, Nath A (2021) Microvascular Injury in the Brains of Patients with Covid-19. N Engl J Med 384:481\\u0026ndash;483\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLopez-Navarro ER, Gutierrez J (2022) Metalloproteinases and their inhibitors in neurological disease. Naunyn Schmiedebergs Arch Pharmacol 395:27\\u0026ndash;38\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLuczo JM, Edwards SJ, Ardipradja K, Suen WW, Au GG, Marsh GA, Godde N, Rootes CL, Bingham J, Sundaramoorthy V (2024) SARS-CoV and SARS-CoV-2 display limited neuronal infection and lack the ability to transmit within synaptically connected axons in stem cell-derived human neurons. J Neurovirol\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMahin A, Soman SP, Modi PK, Raju R, Keshava Prasad TS, Abhinand CS (2024) Meta-analysis of the serum/plasma proteome identifies significant associations between COVID-19 with Alzheimer's/Parkinson's diseases. J Neurovirol\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMeinhardt J, Radke J, Dittmayer C, Franz J, Thomas C, Mothes R, Laue M, Schneider J, Brunink S, Greuel S, Lehmann M, Hassan O, Aschman T, Schumann E, Chua RL, Conrad C, Eils R, Stenzel W, Windgassen M, Rossler L, Goebel HH, Gelderblom HR, Martin H, Nitsche A, Schulz-Schaeffer WJ, Hakroush S, Winkler MS, Tampe B, Scheibe F, Kortvelyessy P, Reinhold D, Siegmund B, Kuhl AA, Elezkurtaj S, Horst D, Oesterhelweg L, Tsokos M, Ingold-Heppner B, Stadelmann C, Drosten C, Corman VM, Radbruch H (2021) and F.L. Heppner. Olfactory transmucosal SARS-CoV-2 invasion as a port of central nervous system entry in individuals with COVID-19. \\u003cem\\u003eNat Neurosci\\u003c/em\\u003e 24:168\\u0026ndash;175\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMurphy SL, Halvorsen B, Holter JC, Huse C, Tveita A, Troseid M, Hoel H, Kildal AB, Holten AR, Lerum TV, Skjonsberg OH, Michelsen AE, Aalokken TM, T.N., Nor-Solidarity Consortium K, Tonby A, Lind S, Dudman BK, Granerud L, Heggelund S, Boe AM, Dyrholt-Riise P, Aukrust A, Barratt-Due T, Ueland, Dahl TB (2023) Circulating markers of extracellular matrix remodelling in severe COVID-19 patients. J Intern Med 294:784\\u0026ndash;797\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eOlajide OA, Iwuanyanwu VU, Adegbola OD, Al-Hindawi AA (2022) SARS-CoV-2 Spike Glycoprotein S1 Induces Neuroinflammation in BV-2 Microglia. Mol Neurobiol 59:445\\u0026ndash;458\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePatterson BK, Francisco EB, Yogendra R, Long E, Pise A, Rodrigues H, Hall E, Herrera M, Parikh P, Guevara-Coto J, Triche TJ, Scott P, Hekmati S, Maglinte D, Chang X, Mora-Rodriguez RA, Mora J (2021) Persistence of SARS CoV-2 S1 Protein in CD16\\u0026thinsp;+\\u0026thinsp;Monocytes in Post-Acute Sequelae of COVID-19 (PASC) up to 15 Months Post-Infection. Front Immunol 12:746021\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePeluso MJ, Deeks SG, Mustapic M, Kapogiannis D, Henrich TJ, Lu S, Goldberg SA, Hoh R, Chen JY, Martinez EO, Kelly JD, Martin JN, Goetzl EJ (2022) SARS-CoV-2 and Mitochondrial Proteins in Neural-Derived Exosomes of COVID-19. Ann Neurol 91:772\\u0026ndash;781\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePerry VH, Nicoll JA, Holmes C (2010) Microglia in neurodegenerative disease. Nat Rev Neurol 6:193\\u0026ndash;201\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePhillips S, Williams MA (2021) Confronting Our Next National Health Disaster - Long-Haul Covid. N Engl J Med 385:577\\u0026ndash;579\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eProal AD, VanElzakker MB (2021) Long COVID or Post-acute Sequelae of COVID-19 (PASC): An Overview of Biological Factors That May Contribute to Persistent Symptoms. Front Microbiol 12:698169\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eProal AD, VanElzakker MB, Aleman S, Bach K, Boribong BP, Buggert M, Cherry S, Chertow DS, Davies HE, Dupont CL, Deeks SG, Eimer W, Ely EW, Fasano A, Freire M, Geng LN, Griffin DE, Henrich TJ, Iwasaki A, Izquierdo-Garcia D, Locci M, Mehandru S, Painter MM, Peluso MJ, Pretorius E, Price DA, Putrino D, Scheuermann RH, Tan GS, Tanzi RE, VanBrocklin HF, Yonker LM, Wherry EJ (2023) Author Correction: SARS-CoV-2 reservoir in post-acute sequelae of COVID-19 (PASC). Nat Immunol 24:1778\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eRempe RG, Hartz AM, Bauer B (2016) Matrix metalloproteinases in the brain and blood-brain barrier: versatile breakers and makers. J Cereb Blood Flow Metab 36:1481\\u0026ndash;14507\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSamudyata AO, Oliveira S, Malwade N, Rufino de Sousa SK, Goparaju J, Gracias F, Orhan L, Steponaviciute M, Schalling SD, Sheridan RH, Perlis AG, Rothfuchs, Sellgren CM (2022) SARS-CoV-2 promotes microglial synapse elimination in human brain organoids. Mol Psychiatry 27:3939\\u0026ndash;3950\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSavic G, Stevanovic I, Mihajlovic D, Jurisevic M, Gajovic N, Jovanovic I, Ninkovic M (2022) MMP-9/BDNF ratio predicts more severe COVID-19 outcomes. Int J Med Sci 19:1903\\u0026ndash;1911\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eShanley JE, Valenciano AF, Timmons G, Miner AE, Kakarla V, Rempe T, Yang JH, Gooding A, Norman MA, Banks SJ, Ritter ML, Ellis RJ, Horton L, Graves JS (2022) Longitudinal evaluation of neurologic-post acute sequelae SARS-CoV-2 infection symptoms. Ann Clin Transl Neurol 9:995\\u0026ndash;1010\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSodagar A, Javed R, Tahir H, Razak SIA, Shakir M, Naeem M, Yusof AHA, Sagadevan S, Hazafa A, Uddin J, Khan A, Al-Harrasi A (2022) Pathological Features and Neuroinflammatory Mechanisms of SARS-CoV-2 in the Brain and Potential Therapeutic Approaches. \\u003cem\\u003eBiomolecules\\u003c/em\\u003e 12\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eStawarski M, Stefaniuk M, Wlodarczyk J (2014) Matrix metalloproteinase-9 involvement in the structural plasticity of dendritic spines. Front Neuroanat 8:68\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSunny A, James RR, Menon SR, Rayaroth S, Daniel A, Thompson NA, Tharakan B (2024) Matrix Metalloproteinase-9 inhibitors as therapeutic drugs for traumatic brain injury. Neurochem Int 172:105642\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSwank Z, Senussi Y, Manickas-Hill Z, Yu XG, Li JZ, Alter G, Walt DR (2023) Persistent Circulating Severe Acute Respiratory Syndrome Coronavirus 2 Spike Is Associated With Post-acute Coronavirus Disease 2019 Sequelae. Clin Infect Dis 76:e487\\u0026ndash;e490\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTai W, He L, Zhang X, Pu J, Voronin D, Jiang S, Zhou Y, Du L (2020) Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol 17:613\\u0026ndash;620\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eThaweethai, T., S.E. Jolley, E.W. Karlson, E.B. Levitan, B. Levy, G.A. McComsey, L.McCorkell, G.N. Nadkarni, S. Parthasarathy, U. Singh, T.A. Walker, C.A. Selvaggi,D.J. Shinnick, C.C.M. Schulte, R. Atchley-Challenner, G.A. Alba, R. Alicic, N. Altman,K. Anglin, U. Argueta, H. Ashktorab, G. Baslet, I.V. Bassett, L. Bateman, B. Bedi,S. Bhattacharyya, M.A. Bind, A.L. Blomkalns, H. Bonilla, P.A. Bush, M. Castro, J.Chan, A.W. Charney, P. Chen, L.B. Chibnik, H.Y. Chu, R.G. Clifton, M.M. Costantine,S.K. Cribbs, S.I. Davila Nieves, S.G. Deeks, A. Duven, I.F. Emery, N. Erdmann, K.M.Erlandson, K.C. Ernst, R. Farah-Abraham, C.E. Farner, E.M. Feuerriegel, J. Fleurimont,V. Fonseca, N. Franko, V. Gainer, J.C. Gander, E.M. Gardner, L.N. Geng, K.S. Gibson,M. Go, J.D. Goldman, H. Grebe, F.L. Greenway, M. Habli, J. Hafner, J.E. Han, K.A.Hanson, J. Heath, C. Hernandez, R. Hess, S.L. Hodder, M.K. Hoffman, S.E. Hoover, B.Huang, B.L. Hughes, P. Jagannathan, J. John, M.R. Jordan, S.D. Katz, E.S. Kaufman,J.D. Kelly, S.W. Kelly, M.M. Kemp, J.P. Kirwan, J.D. Klein, K.S. Knox, J.A. Krishnan,A. Kumar, A.O. Laiyemo, A.A. Lambert, M. Lanca, J.K. Lee-Iannotti, B.P. Logarbo, M.T.Longo, C.A. Luciano, K. Lutrick, J.H. Maley, J.G. Marathe, V. Marconi, G.D. Marshall,C.F. Martin, Y. Matusov, A. Mehari, H. Mendez-Figueroa, R. Mermelstein, T.D. Metz,R. Morse, J. Mosier, C. Mouchati, J. Mullington, S.N. Murphy, R.B. Neuman, J.Z. Nikolich,I. Ofotokun, E. Ojemakinde, A. Palatnik, K. Palomares, T. Parimon, S. Parry, J.E.Patterson, T.F. Patterson, R.E. Patzer, M.J. Peluso, P. Pemu, C.M. Pettker, B.A. Plunkett,K. Pogreba-Brown, A. Poppas, J.G. Quigley, U. Reddy, R. Reece, H. Reeder, W.B. Reeves,E.M. Reiman, F. Rischard, J. Rosand, D.J. Rouse, A. Ruff, G. Saade, G.J. Sandoval,S.M. Schlater, F. Shepherd, Z.A. Sherif, H. Simhan, N.G. Singer, D.W. Skupski, A.Sowles, J.A. Sparks, F.I. Sukhera, B.S. Taylor, L. Teunis, R.J. Thomas, J.M. Thorp,P. Thuluvath, A. Ticotsky, A.T. Tita, K.R. Tuttle, A.E. Urdaneta, D. Valdivieso, T.M.VanWagoner, A. Vasey, M. Verduzco-Gutierrez, Z.S. Wallace, H.D. Ward, D.E. Warren,S.J. Weiner, S. Welch, S.W. Whiteheart, Z. Wiley, J.P. Wisnivesky, L.M. Yee, S. Zisis,L.I. Horwitz, A.S. Foulkes, and R. Consortium. 2023. Development of a Definition of Postacute Sequelae of SARS-CoV-2 Infection. \\u003cem\\u003eJAMA\\u003c/em\\u003e 329:1934\\u0026ndash;1946\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTheoharides TC, Kempuraj D (2023) Role of SARS-CoV-2 Spike-Protein-Induced Activation of Microglia and Mast Cells in the Pathogenesis of Neuro-COVID. Cells 12:688\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTremblay ME, Madore C, Tian L, Verkhratsky A (2022) Editorial: Role of Neuroinflammation in the Neuropsychiatric and Neurological Aspects of COVID-19. Front Cell Neurosci 16:840121\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTsilioni I, Theoharides TC (2023) Recombinant SARS-CoV-2 Spike Protein and Its Receptor Binding Domain Stimulate Release of Different Pro-Inflammatory Mediators via Activation of Distinct Receptors on Human Microglia Cells. Mol Neurobiol\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWierzba-Bobrowicz T, Krajewski P, Tarka S, Acewicz A, Felczak P, Stepien T, M PG, Grzegorczyk M (2021) Neuropathological analysis of the brains of fifty-two patients with COVID-19. Folia Neuropathol 59:219\\u0026ndash;231\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eXu Z, Wang H, Jiang S, Teng J, Zhou D, Chen Z, Wen C, Xu Z (2024) Brain Pathology in COVID-19: Clinical Manifestations and Potential Mechanisms. Neurosci Bull 40:383\\u0026ndash;400\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYang AC, Kern F, Losada PM, Agam MR, Maat CA, Schmartz GP, Fehlmann T, Stein JA, Schaum N, Lee DP, Calcuttawala K, Vest RT, Berdnik D, Lu N, Hahn O, Gate D, McNerney MW, Channappa D, Cobos I, Ludwig N, Schulz-Schaeffer WJ, Keller A, Wyss-Coray T (2021) Dysregulation of brain and choroid plexus cell types in severe COVID-19. Nature 595:565\\u0026ndash;571\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZingaropoli MA, Iannetta M, Piermatteo L, Pasculli P, Latronico T, Mazzuti L, Campogiani L, Duca L, Ferraguti G, De Michele M, Galardo G, Pugliese F, Antonelli G, Andreoni M, Sarmati L, Lichtner M, Turriziani O, Ceccherini-Silberstein F, Liuzzi GM, Mastroianni CM, Ciardi MR (2022) Neuro-Axonal Damage and Alteration of Blood-Brain Barrier Integrity in COVID-19 Patients. Cells 11:2480\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"brain, inflammation, Long COVID, mast cells, microglia, MMP-9\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4151696/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4151696/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eLong-COVID is a major health concern because many patients develop chronic neuropsychiatric symptoms, but the precise pathogenesis is unknown. Matrix metalloproteinase-9 (MMP-9) can disrupt neuronal connectivity and was elevated in patients with COVID-19. MMP-9 was measured in the serum of long COVID patients and healthy controls, as well as in the supernatant fluid of cultured human SV-40 microglia, by commercial ELISA. Results were analyzed with one-way ANOVA. MMP-9 in the serum of Long-COVID patients and supernatant fluid from cultured human microglia stimulated by recombinant SARS-CoV-2 Spike protein was assayed by ELISA. MMP-9 was significantly elevated in the serum of Long-COVID patients compared to healthy controls. Moreover, cultured human microglia released MMP-9 when stimulated by Spike protein.\\u003c/p\\u003e \\u003cp\\u003eIn conclusion, MMP-9 may contribute to the development of Long-COVID and serve both as a prognostic biomarker and as target for treatment.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Increased Serum MMP-9 in Long-COVID May Reflect Activation of Microglia by SARS-CoV-2 Spike Protein\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-04-01 16:35:13\",\"doi\":\"10.21203/rs.3.rs-4151696/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"1f231ba1-b790-4715-8337-63997ddd4557\",\"owner\":[],\"postedDate\":\"April 1st, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-04-18T20:02:28+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-04-01 16:35:13\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4151696\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4151696\",\"identity\":\"rs-4151696\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}