RESSALVA Atendendo solicitação do(a) autor(a), o texto completo desta dissertação será disponibilizado somente a partir de 23/03/2025. UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE MEDICINA VETERINÁRIA E ZOOTECNIA Priming com INF-Ƴ e TNF-α aumenta potencial imunomodulador de células tronco mesenquimais caninas in vitro NATIELLY DIAS CHIMENES BOTUCATU – SÃO PAULO MARÇO – 2023 UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE MEDICINA VETERINÁRIA E ZOOTECNIA Priming com INF-Ƴ e TNF-α aumenta potencial imunomodulador de células tronco mesenquimais caninas in vitro NATIELLY DIAS CHIMENES BOTUCATU-SÃO PAULO MARÇO 2023 Dissertação apresentada ao curso de Pós- graduação em Medicina Veterinária, como requisito para elaboração da dissertação para obtenção do título de Mestre. Orientador: Prof. Associado Rogério Martins Amorim ii iii TÍTULO: Priming com INF-Ƴ e TNF-α aumenta potencial imunomodulador de células tronco mesenquimais caninas in vitro COMISSÃO EXAMINADORA __________________________________ Prof. Dr. Rogério Martins Amorim Presidente e Orientador Departamento de Clínica Veterinária FMVZ – UNESP – Botucatu, são Paulo. __________________________________ Prof. Dr. Alexandre Leite Rodrigues de Oliveira Membro da Banca Departamento de Biologia Estrutural e Funcional/ Laboratório de Regeneração Nervosa - UNICAMP __________________________________ Profa. Dra Clelia Akiko Hiruma Lima Membro da Banca Departamento de Biologia Estrutural e Funcional/ INSTITUTO DE BIOCIÊNCIAS- UNESP/Botucatu Data da defesa: 23 de março de 2023. iv AGRADECIMENTOS À minha família pelo suporte ao longo de todo o período de mestrado, em especial ao meu esposo Wislei Luiz Delmondes Taira, sem vocês não seria possível a mudança de cidade, as viagens mensais, os cuidados com os meus cachorros e a realização de todo o projeto de pesquisa. Agradeço ao meu orientador, Prof. Dr. Rogério Martins Amorim, pela orientação e ensinamentos ao longo de todo mestrado, pela confiança depositada a mim para a condução dessa pesquisa, por me receber como parte da equipe em um momento tão difícil com o fim da pandemia mesmo sem me conhecer e por dar todo o suporte diante dos deafios enfrentados. Aos meus colegas e amigos Beatriz da Costa Kamura, Lucas Vinícius de Oliveira Ferreira e João Pedro Marmol de Oliveira, pelo apoio, incentivo e toda a ajuda que foi essencial para a realização de cada parte deste projeto, vocês se tornaram minha família em Botucatu e fizeram eu me sentir em casa, me abraçaram como parte da equipe de pesquisa de uma forma muito acolhedora, sempre um ajudando o outro sem que nenhuma palavra precisasse ser dita. Ao aluno de iniciação científica Paulo Cézar Leão Eliam por todo o apoio e suporte ao longo dos últimos meses de realização do projeto, foi você quem esteve presente nos momentos mais difíceis da pequisa. A Dra. Giovana Boff Sánchez por todo o auxílio, discussão e ajuda a distância para a realização da metodologia do projeto. A Cristiana Raach Bromberger que foi meu suporte em casa, me ouvindo, me apoiando e me ajudando a superar cada obstáculo ao longo desse período e a Andresa Xavier Frade por ter sido meu suporte mesmo a distância no ínicio do mestrado quando nem no laboratória era possível ir por causa da pandemia, superamos juntas as estapas do mestrado. v O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) - Código de Financiamento 001. Obrigada. vi LISTA DE TABELAS Tabela 1. Oligonucleotídeos iniciadores caninos utilizados no qPCR..............64-65 vii LISTA DE APÊNDICES Apêndice 1. Imagem de Ressonância Magnética do encéfalo do animal 1 demonstrando as lesões causadas pela MUO........................................................97 Apêndice 2. Imagem de Ressonância Magnética do encéfalo do animal 2 demonstrando as lesões causadas pela MUO........................................................98 Apêndice 3. Imagem de Ressonância Magnética do encéfalo do animal 3 demonstrando as lesões causadas pela MUO........................................................99 Apêndice 4. Análise do LCR dos animais 1, 2 e 3.................................................99 Apêndice 5. Viabilidade e apoptose das Ad-MSCs..............................................100 Apêndice 6. Atividade metabólica celular (%) de grupos experimentais tratados com LCR canino nas concentrações de 5%, 10%, 15% e 20% (A), e com soro canino nas concentrações de 5%, 10%, 15% e 20% (B).........................................................101 Apêndice 7. Análise descritiva de amostras de LCR (n=1) e soro (n=2) de cães saudáveis em comparação com o LCR (n=3) e soro (n=1) de cães com MUO usados no priming de cAd-MSCs.......................................................................................102 Apêndice 8. Análise estatística da expressão gênica relativa de BNDF, GNDF, HGF, IDO, INF-Ƴ, TNF-α, PTGE2 e IL-10 no priming das cAd-MSCs..................103 viii LISTA DE FIGURAS Figura 1. Esquema representando as influências das citocinas pró e anti- inflamatórias sobre a micróglia (adaptado de COATES;JEFFERY, 2014).......................................................................................................................25 Figura 2. Esquema demonstrando as possibilidades de técnica priming (adaptado de LI et al., 2022) ...................................................................................................39 Figura 3. Scheme represents MUO and MS neuroinflammation............................55 Figura 4. Experimental design................................................................................58 Figura 5. cAd-MSC immunophenotypic analysis (AH), cAd-MSC viability and apoptosis (IK) and cAd-MSC ability to differentiate (LO).........................................................................................................................68 Figura 6. Control and priming with INF-γ and TNF-α of cAd-MSCs. .....................71 Figura 7. cAd-MSC priming with healthy and MUO dog plasma…………...............72 Figura 8. cAd-MSCs priming with CSF from healthy dogs and MUO………...........73 Figura 9. Post- priming cAd-MSC viability..............................................................74 Figura 10. Relative gene expression of BNDF, GNDF, HGF, IDO, INF-Ƴ, TNF-α, PTGE2 and IL-10 in the priming groups of cAd-MSCs.............................................76 Figura 11. Secretion profile of IL-8, IL-10, GM-CSF, IL-2 and MCP-1 in the cAd- MSC priming groups................................................................................................78 ix LISTA DE ABREVIAÇÕES Ad-MSCs- Células Tronco Mesenquimais derivadas do tecido adiposo BDNF- fator neurotrófico derivado do cérebro Breg- célula B reguladora cAMP- AMP cíclico C- grupo experimental controle cAd-MSCs- Células Tronco Mesenquimais derivadas do tecido adiposo canina CCL2- ligante de quimiocina 2 CD- células dendríticas C-LCR- grupo experimental com líquido cefalorraquidiano de cão crontrole COX 1- ciclo-oxigenase 1 COX 2- ciclo-oxigenase 2 C-S- grupo experimental com soro de cão controle CXCL8- interleucina 8 cDNA- ácido desoxirribonucleico complementar DLA classe II- dog leucocyte antingen classe II DMEM- Dulbecco's Modified Eagle's Medium DMSO- dimetilsufóxido DPBS- Dubecco’s Phosphate Buffered Saline EAE- encefalite experimental autoimune EM- esclerose múltipla GCN2- controle geral não depressível 2 x GDNF-fator neurotrófico derivado das células da glia GM-CSF- fator estimulador de colônias de granulócitos e macrófagos GVHD- doença do enxerto contra o hospedeiro HGF- fator de crescimento de hepatócitos HLA-DR- molécula do complexo de histocompatibilidade de classe II HLA-G- antígeno leucocitário humano G HO-1- heme oxigenase 1 HPRT- hipoxantina-guanina fosforribosiltransferase IDO- indoleamina -2,3- dioxigenase IF2- fator de iniciação da tradução eucariótica IFN-Ƴ- interferon gama IL-1- interleucina 1 IL-1α- interleucina 1 alfa IL-1ß- interleucina 1 beta IL1RA- antagonista de receptor de interleucina 1 IL-4- interleucina 4 IL-5- interleucina 5 IL-6- interleucina 6 IL-8- interleucina 8 IL-10- interleucina 10 IL-12- interleucina 12 IL-13- interleucina 13 IL-17- interleucina 17 xi IL-23- interleucina 23 IL-35- interleucina 35 iNOS- óxido nítrico sintase induzida LCR-líquido cefalorraquidiano LEN- leucoencefalite necrosante LPS- lipopolissacarídeo M1- fenótipo da micróglia pró-inflamatório M2- fenótipo da micróglia anti-inflamatório MCP-1- proteína quimioatraente de monócitos 1 MEG- meningoencefalomielite granulomatosa MEN- meningoencefalite necrosante MHC II- molécula do complexo de histocompatibilidade de classe II mRNA- ácido ribonucleico mensageiro MSCs- células tronco mesenquimais MUO- meningoencefalite de origem desconhecida MUO-LCR- grupo experimental com liquido cefalorraquidiano de cão com MUO MUO-S- grupo experimental com soro de cão com MUO MTT- (3-(4,5-dimetiltiazol-2yl)-2,5-difenil brometo de tetrazolina) NF-kβ- fator nuclear kappa beta NGF- fator de crescimento neural NK- natural killer NO- óxido nítrico NOS- óxido nítrico sintase xii P0- passagem celular zero P1- passagem celular 1 P2- passagem celular 2 P3- passagem celular 3 P4- passagem celular 4 PD-L1- ligante de morte 1 programado PD-L2- ligante de morte 2 programado PGE2- prostaglandina E 2 PTGES2- prostaglandina E sintetase 2 qPCR- reação em cadeia de polimerase quantitativa em tempo real RNA- ácido ribonucleico RM- ressonância magnética ROS- espécies reativas de oxigênio RPS5- proteína ribossômica S5 RPS19- proteína ribossômica S19 SFB- soro fetal bovino SNC- sistema nervoso central TGF-ß- fator de crescimento transformador beta Th- linfócito T helper TNF-α- fator de necrose tumoral beta Treg- linfócitos T reguladores TRLs- receptor tipo toll like TSG-6- proteína do gene 6 induzida xiii SUMÁRIO Página RESUMO .............................................................................................................. xiv Abstract .................................................................................................................. xv 1 INTRODUÇÃO ................................................................................................... 17 1.1 REVISÃO DE LITERATURA ........................................................................... 22 1.1.1 Meningoencefalite de origem desconhecida (MUO) ................................. 22 1.1.2 Etiopatogenia ....................................................................................... 23 1.1.3 Diagnóstico, tratamento e prognóstico ................................................ 26 1.1. 4 Esclerose múltipla (EM) ........................................................................... 28 1.1. 5 Células Estromais/ Tronco Mesenquimais Multipotentes (MSCs) ........... 30 1.1. 6 MSCs e Neuroinflamação ........................................................................ 31 1.1.7 Citocinas e Quimiocinas Pró-inflamatórias ............................................... 32 1.1.8 Citocinas Anti-inflamatórias ...................................................................... 35 1.1.9 Fatores neurotróficos ................................................................................ 36 1.1.10 Prostaglandina E 2 (PGE2) ..................................................................... 38 1.1. 11 Priming de MSCs ................................................................................... 38 Objetivos ............................................................................................................... 42 BIBLIOGRAFIA ..................................................................................................... 43 2.TRABALHO CIENTÍFICO................................................................................... 53 3 CONCLUSÃO GERAL ....................................................................................... 95 APÊNDICE ........................................................................................................ 96 CHIMENES, N.C. Priming com INF-Ƴ e TNF-α aumenta potencial imunomodulador de células tronco mesenquimais caninas in vitro. Botucatu, 2023. 103 p. Defesa (Mestrado) – Faculdade de Medicina Veterinária e Zootecnia, Campus de Botucatu, Universidade Estadual Paulista. RESUMO A meningoencefalite de origem desconhecida (MUO) é uma enfermidade neuroimune em cães, com grande potencial de se tornar um modelo animal natural para a esclerose múltipla. O priming in vitro das células tronco mesenquimais derivadas de tecido adiposo canino (cAd-MSCs) é uma estratégia para potencializar a ação imunomodulatória e imunossupressora das cAd-MSCs. O objetivo deste trabalho foi avaliar o priming com IFN-Ƴ e TNF-α, LCR e soro de cães com MUO nas cAd-MSCs. Para tanto, foi realizado o priming in vitro por 72 horas das cAd- MSCs (P3-P4) em seis grupos: controle (C), com IFN-Ƴ em associação com TNF-α (IFN-Ƴ+ TNF-α), soro controle (C-S), soro de cães com MUO (MUO-S), LCR controle (C-LCR) e LCR de cães com MUO (MUO-LCR) e avaliada a expressão gênica para BDNF, GNDF, HGF, IDO, PTGE2, IL-10, IFN-Ƴ e TNF-α por qPCR e quantificação proteica de GM-CSF, IL-10, IL-2, IL-8 e MCP-1 por ensaio multiplexado. Comparando todos os grupos com o grupo C, o IFN-Ƴ+ TNF-α apresentou aumento de HGF, IDO e TNF-α e IL-8 (p= <0,001), nos grupos C-LCR e MUO-LCR houve aumento de IFN-Ƴ e TNF-α e redução de BDNF e GNDF (p= <0,001), os grupos C-S e MUO-S apresentaram redução na quantificação proteica de IL-10 (p= <0,001), e o grupo C-S teve redução de BDNF e GNDF (p= <0,001) e o grupo MUO-S teve redução de HGF (p= <0,001). Diante dos resultados, a técnica priming IFN-Ƴ + TNF-α demonstrou maior potencial de perfil imunomodulatório com tendência anti-inflamatória em comparação com os demais grupos. Palavras-chaves: neuroimunes, citocinas, meningoencefalite, esclerose múltipla. CHIMENES, N.C. Priming INF-α and TNF-α enhances the immunomodulatory potential of canine mesenchymal stem cells in vitro. Botucatu, 2023. 103 p. Defesa (Mestrado) – Faculdade de Medicina Veterinária e Zootecnia, Campus de Botucatu, Universidade Estadual Paulista. Abstract Meningoencephalitis of unknown origin (MUO) is a neuroimmune disease affects dogs, with great potential to become a natural experimental model for multiple sclerosis. In vitro priming of mesenchymal stem cells derived from canine adipose tissue (cAd-MSCs) is a strategy to enhances the immunomodulatory and immunosuppressive action of cAd-MSCs. The aim of this work is to evaluate the priming with IFN-γ and TNF-α, CSF and serum from dogs with MUO on cAd-MSCs. Therefore, in vitro priming of cAd-MSCs was performed in six groups: control (C), with IFN-α in association with TNF-α (IFN-α + TNF-α), control serum (C-P), serum from dogs with MUO (MUO-P), CSF control (C-CSF) and CSF from dogs with MUO (MUO-CSF) and evaluated gene expression for BDNF, GNDF, HGF, IDO, PTGE2, IL-10, IFN- Ƴ and TNF-α by qPCR and protein expression of GM-CSF, IL-10, IL-2, IL-8 and MCP-1 by multiplex assay. In comparison with group C, the group with IFN- γ+ TNF-α showed an increase in HGF, IDO and TNF-α and IL-8 (p= <0.001), in the C-CSF and MUO-CSF groups there was an increase in IFN -Ƴ and TNF-α and a reduction in BDNF and GNDF (p= <0.001), the C-S and MUO-S groups showed a reduction in IL-10 (p= <0.001), and the C-S group had a reduction in BDNF and GNDF ( p= <0.001) and the MUO-S group had reduced HGF (p= <0.001). In view of the results, the IFN-γ + TNF-α priming technique demonstrated a greater potencial for immunomodulatory profile with an anti-inflmmatory tendency compared to the other groups. Keywords: neuroimmune, cytokines, meningoencephalitis, multiple sclerosis. 43 BIBLIOGRAFIA ABBAS, A. K. et al. Revisiting IL-2: Biology and therapeutic prospects. Science Immunology, v. 3, n. 25, 2018. ALAGESAN, S. et al. Enhancement strategies for mesenchymal stem cells and related therapies. Stem Cell Research and Therapy, v. 13, n. 1, p. 1–16, 2022. ALDHSHAN, M. S. et al. Glucose Stimulates Glial Cell Line-Derived Neurotrophic Factor Gene Expression in Microglia through a GLUT5-Independent Mechanism. International Journal of Molecular Sciences, v. 23, n. 13, p. 1–13, 2022. AMORIM, R. M. et al. Placenta-derived multipotent mesenchymal stromal cells: A promising potential cell-based therapy for canine inflammatory brain disease. Stem Cell Research and Therapy, v. 11, n. 1, p. 1–12, 2020. ANDERSEN-RANBERG, E.; BERENDT, M.; GREDAL, H. Biomarkers of non- infectious inflammatory CNS diseases in dogs — Where are we now? Part I: Meningoencephalitis of unknown origin. Veterinary Journal, v. 273, p. 105678, 2021. ANDRZEJEWSKA, A.; LUKOMSKA, B.; JANOWSKI, M. Concise Review: Mesenchymal Stem Cells: From Roots to Boost. Stem Cells, v. 37, n. 7, p. 855–864, 2019. BAKER, D.; AMOR, S. Experimental autoimmune encephalomyelitis is a good model of multiple sclerosis if used wisely. Multiple Sclerosis and Related Disorders, v. 3, n. 5, p. 555–564, 2014. BARBER, R. M. et al. Identification of risk loci for necrotizing meningoencephalitis in Pug dogs. The Journal of heredity, v. 102 Suppl, p. 40–46, 2011. BECKMANN, K. et al. A newly designed radiation therapy protocol in combination with prednisolone as treatment for meningoencephalitis of unknown origin in dogs: A prospective pilot study introducing magnetic resonance spectroscopy as monitor tool. Acta Veterinaria Scandinavica, v. 57, n. 1, 2015. 44 BENKHOUCHA, M. et al. Hepatocyte growth factor inhibits CNS autoimmunity by inducing tolerogenic dendritic cells and CD25+Foxp3+ regulatory T cells. Proceedings of the National Academy of Sciences of the United States of America, v. 107, n. 14, p. 6424–6429, 2010. CARRADE, D. D.; BORJESSON, D. L. Immunomodulation by mesenchymal stem cells in veterinary species. Comparative Medicine, v. 63, n. 3, p. 207–217, 2013. CHEN, P.-M. et al. Induction of immunomodulatory monocytes by human mesenchymal stem cell-derived hepatocyte growth factor through ERK1/2. Journal of Leukocyte Biology, v. 96, n. 2, p. 295–303, 2014. CHEN, X.; WANG, S.; CAO, W. Mesenchymal stem cell-mediated immunomodulation in cell therapy of neurodegenerative diseases. Cellular Immunology, v. 326, p. 8–14, 2018. COATES, J. R.; JEFFERY, N. D. Perspectives on meningoencephalomyelitis of unknown origin. Veterinary Clinics of North America - Small Animal Practice, v. 44, n. 6, p. 1157–1185, 2014. COLUCCI-D’AMATO, L.; SPERANZA, L.; VOLPICELLI, F. Neurotrophic factor bdnf, physiological functions and therapeutic potential in depression, neurodegeneration and brain cancer. International Journal of Molecular Sciences, v. 21, n. 20, p. 1– 29, 2020. CONG, H. et al. Icariin ameliorates the progression of experimental autoimmune encephalomyelitis by down-regulating the major inflammatory signal pathways in a mouse relapse-remission model of multiple sclerosis. European Journal of Pharmacology, v. 885, n. May, 2020. CONSTANTINESCU, C. S. et al. Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). British Journal of Pharmacology, v. 164, n. 4, p. 1079–1106, 2011. COOPER, J. J. et al. Necrotizing meningoencephalitis in atypical dog breeds: A case series and literature review. Journal of Veterinary Internal Medicine, v. 28, n. 1, 45 p. 198–203, 2014. CORNELIS, I. et al. Clinical presentation, diagnostic findings, prognostic factors, treatment and outcome in dogs with meningoencephalomyelitis of unknown origin: A review. Veterinary Journal, v. 244, p. 37–44, 2019. CORNELIS, I.; VOLK, H. A.; DE DECKER, S. Clinical presentation, diagnostic findings and long-term survival in large breed dogs with meningoencephalitis of unknown aetiology. Veterinary Record, v. 179, n. 6, 2016. DA GAMA PEREIRA, A. B. C. N. et al. Prevalence of multiple sclerosis in Brazil: A systematic review. Multiple Sclerosis and Related Disorders, v. 4, n. 6, p. 572– 579, 2015. DENDROU, C. A.; FUGGER, L.; FRIESE, M. A. Immunopathology of multiple sclerosis. Nature Reviews Immunology, v. 15, n. 9, p. 545–558, 2015. DOSHI, A.; CHATAWAY, J. Doshi2016. v. 16, n. 6, p. 53–59, 2016. EBERLY, J. A. et al. Pathology in Practice. n. D, p. 361–363, [s.d.]. FARIVAR, S. et al. Neural differentiation of human umbilical cord mesenchymal stem cells by cerebrospinal fluid. Iranian Journal of Child Neurology, v. 9, n. 1, p. 87– 93, 2015. GAO, F. et al. Mesenchymal stem cells and immunomodulation: Current status and future prospects. Cell Death and Disease, v. 7, n. 1, 2016. GENC, B. et al. Stem cell therapy for multiple sclerosis. Cochrane Database of Systematic Reviews, v. 2019, n. 9, 2019. GONÇALVES, R. et al. Inflammatory Disease Affecting the Central Nervous System in Dogs: A Retrospective Study in England (2010–2019). Frontiers in Veterinary Science, v. 8, n. January, p. 1–11, 2022. GRANGER, N. et al. Clinical findings and treatment of non-infectious meningoencephalomyelitis in dogs: A systematic review of 457 published cases from 1962 to 2008. Veterinary Journal, v. 44, n. 3, p. 290–297, 2010. 46 GRANGER, N.; SMITH, P. M.; JEFFERY, N. D. Clinical findings and treatment of non-infectious meningoencephalomyelitis in dogs: A systematic review of 457 published cases from 1962 to 2008. Veterinary Journal, v. 184, n. 3, p. 290–297, 2010. GREER, K. A. et al. Necrotizing meningoencephalitis of Pug Dogs associates with dog leukocyte antigen class II and resembles acute variant forms of multiple sclerosis. Tissue Antigens, v. 76, n. 2, p. 110–118, 2010. GUTIÉRREZ, I. L. et al. CCL2 Inhibition of Pro-Resolving Mediators Potentiates Neuroinflammation in Astrocytes. International Journal of Molecular Sciences, v. 23, n. 6, 2022. HA, H.; DEBNATH, B.; NEAMATI, N. Role of the CXCL8-CXCR1/2 axis in cancer and inflammatory diseases. Theranostics, v. 7, n. 6, p. 1543–1588, 2017. HAMILTON, J. A. Cytokines Focus GM-CSF in inflammation. Journal of Experimental Medicine, v. 217, n. 1, p. 1–16, 2019. HARRIS, V. K. et al. Clinical and pathological effects of intrathecal injection of mesenchymal stem cell-derived neural progenitors in an experimental model of multiple sclerosis. Journal of the Neurological Sciences, v. 313, n. 1–2, p. 167– 177, 2012. HOFFMAN, A. M.; DOWB, S. W. Machine Translated by Google TRADUCIONAL E CLÍNICA Revisão Concisa : Testes com Células-Tronco Usando Modelos de Doenças de Animais de Companhia Machine Translated by Google. p. 1709–1729, 2016. HU, W.; LUCCHINETTI, C. F. The pathological spectrum of CNS inflammatory demyelinating diseases. Seminars in Immunopathology, v. 31, n. 4, p. 439–453, 2009. IRIARTE, J.; KATSAMAKIS, G.; CASTRO, P. DE. Multiple Sclerosis multiple sclerosis. v. 51, n. January 2010, p. 169–180, 1999. JIANG, W.; XU, J. Immune modulation by mesenchymal stem cells. Cell 47 Proliferation, v. 53, n. 1, p. 1–16, 2020. KAUR, S. et al. A panoramic review of IL-6: Structure, pathophysiological roles and inhibitors. Bioorganic and Medicinal Chemistry, v. 28, n. 5, p. 115327, 2020. KIM, D. S. et al. Enhanced Immunosuppressive Properties of Human Mesenchymal Stem Cells Primed by Interferon-γ. EBioMedicine, v. 28, p. 261–273, 2018. KLINEOVA, S.; LUBLIN, F. D. Clinical course of multiple sclerosis. Cold Spring Harbor Perspectives in Medicine, v. 8, n. 9, p. 1–12, 2018. LEE, B. C.; KANG, K. S. Functional enhancement strategies for immunomodulation of mesenchymal stem cells and their therapeutic application. Stem Cell Research and Therapy, v. 11, n. 1, p. 1–10, 2020. LEE, J. et al. Cerebrospinal fluid from Alzheimer’s disease patients as an optimal formulation for therapeutic application of mesenchymal stem cells in Alzheimer’s disease. Scientific Reports, v. 9, n. 1, p. 1–9, 2019. LEIJS, M. J. C. et al. Effect of arthritic synovial fluids on the expression of immunomodulatory factors by mesenchymal stem cells: An explorative in vitro study. Frontiers in Immunology, v. 3, n. AUG, p. 1–7, 2012. LI, M. et al. Potential pre-activation strategies for improving therapeutic efficacy of mesenchymal stem cells: current status and future prospects. Stem Cell Research and Therapy, v. 13, n. 1, p. 1–21, 2022. LI, N.; HUA, J. Interactions between mesenchymal stem cells and the immune system. Cellular and Molecular Life Sciences, v. 74, n. 13, p. 2345–2360, 2017. LIMA GIACOBBO, B. et al. Brain-Derived Neurotrophic Factor in Brain Disorders: Focus on Neuroinflammation. Molecular Neurobiology, v. 56, n. 5, p. 3295–3312, 2019. LOWRIE, M.; SMITH, P. M.; GAROSI, L. Meningoencephalitis of unknown origin: investigation of prognostic factors and outcome using a standard treatment protocol. The Veterinary record, v. 172, n. 20, p. 527, 2013. 48 MCGINLEY MP, GOLDSCHMIDT CH, R.-G. A. Diagnosis and Treatment of Multiple Sclerosis: A Review. Journal of the American Medical Association, v. 325(, n. 8, p. 765–779, 2021. MEAD, B. et al. TNFα-mediated priming of mesenchymal stem cells enhances their neuroprotective effect on retinal ganglion cells. Investigative Ophthalmology and Visual Science, v. 61, n. 2, 2020. MIZUI, M. Natural and modified IL-2 for the treatment of cancer and autoimmune diseases. Clinical Immunology, v. 206, p. 63–70, 2019. MUSCARI, C. et al. Priming adult stem cells by hypoxic pretreatments for applications in regenerative medicine. Journal of Biomedical Science, v. 20, n. 1, p. 1–13, 2013. NESSLER, J. N. et al. Canine Meningoencephalitis of Unknown Origin—The Search for Infectious Agents in the Cerebrospinal Fluid via Deep Sequencing. Frontiers in Veterinary Science, v. 8, n. December, p. 1–6, 2021. NORONHA, N. D. C. et al. Correction to: Priming approaches to improve the efficacy of mesenchymal stromal cell-based therapies (Stem Cell Research and Therapy (2019) 10 (131) DOI: 10.1186/s13287-019-1224-y). Stem Cell Research and Therapy, v. 10, n. 1, p. 1–21, 2019. OH, J.; VIDAL-JORDANA, A.; MONTALBAN, X. Multiple sclerosis: Clinical aspects. Current Opinion in Neurology, v. 31, n. 6, p. 752–759, 2018. PARK, E. S.; UCHIDA, K.; NAKAYAMA, H. Comprehensive Immunohistochemical Studies on Canine Necrotizing Meningoencephalitis (NME), Necrotizing Leukoencephalitis (NLE), and Granulomatous Meningoencephalomyelitis (GME). Veterinary Pathology, v. 49, n. 4, p. 682–692, 2012. PARK, E. S.; UCHIDA, K.; NAKAYAMA, H. Th1-, Th2-, and Th17-Related Cytokine and Chemokine Receptor mRNA and Protein Expression in the Brain Tissues, T Cells, and Macrophages of Dogs With Necrotizing and Granulomatous Meningoencephalitis. Veterinary Pathology, v. 50, n. 6, p. 1127–1134, 2013. 49 PERONI, J. F.; BORJESSON, D. L. Anti-Inflammatory and Immunomodulatory Activities of Stem Cells. Veterinary Clinics of North America - Equine Practice, v. 27, n. 2, p. 351–362, 2011. PORTERO, M. et al. Cerebrospinal fluid and blood lactate concentrations as prognostic biomarkers in dogs with meningoencephalitis of unknown origin. Veterinary Journal, v. 254, 2019. PRINZ, M.; JUNG, S.; PRILLER, J. Microglia Biology: One Century of Evolving Concepts. Cell, v. 179, n. 2, p. 292–311, 2019. PROCACCINI, C. et al. Animal models of Multiple Sclerosis. European Journal of Pharmacology, v. 759, p. 182–191, 2015. SAND, I. K. Classification, diagnosis, and differential diagnosis of multiple sclerosis. Current Opinion in Neurology, v. 28, n. 3, p. 193–205, 2015. SARAIVA, M.; O’GARRA, A. The regulation of IL-10 production by immune cells. Nature Reviews Immunology, v. 10, n. 3, p. 170–181, 2010. SCHRAUWEN, I. et al. Identification of novel genetic risk loci in maltese dogs with necrotizing meningoencephalitis and evidence of a shared genetic risk across toy dog breeds. PLoS ONE, v. 9, n. 11, 2014. SILVA-CARVALHO, A. É. et al. GVHD-derived plasma as a priming strategy of mesenchymal stem cells. Stem Cell Research and Therapy, v. 11, n. 1, p. 1–12, 2020. SINGH, S.; ANSHITA, D.; RAVICHANDIRAN, V. MCP-1: Function, regulation, and involvement in disease. International Immunopharmacology, v. 101, p. 107598, 2021. SOSPEDRA, M.; MARTIN, R.; 2016. Immunology of multiple sclerosis. Current Allergy and Asthma Reports, v. 7, n. 4, p. 285–292, 2007. STEE, K. et al. Cytosine arabinoside constant rate infusion without subsequent subcutaneous injections for the treatment of dogs with meningoencephalomyelitis of 50 unknown origin. Veterinary Record, v. 187, n. 11, p. 98, 2020. SUZUMURA, A. Neuron-microglia interactions in neuroinflammation. Clinical and Experimental Neuroimmunology, v. 6, n. 3, p. 225–231, 2015. TALARICO, L. R.; SCHATZBERG, S. J. Idiopathic granulomatous and necrotising inflammatory disorders of the canine central nervous system: A review and future perspectives. Journal of Small Animal Practice, v. 51, n. 3, p. 138–149, 2010. TAN, P. H. et al. Interferons in Pain and Infections: Emerging Roles in Neuro- Immune and Neuro-Glial Interactions. Frontiers in Immunology, v. 12, n. November, p. 1–14, 2021. TANAKA, T.; NARAZAKI, M.; KISHIMOTO, T. patterns (DAMPs), which are released from damaged or dying cells in noninfectious inflammations such as burn or trauma, directly or indirectly promote inflammation. During sterile surgical operations, an increase in serum IL66 levels precedes elevation of. v. 6, n. Kishimoto 1989, p. 1– 16, 2014. THE LANCET NEUROLOGY. Multiple sclerosis under the spotlight. The Lancet Neurology, v. 20, n. 7, p. 497, 2021. TORRE-FUENTES, L. et al. Experimental models of demyelination and remyelination. Neurologia, v. 35, n. 1, p. 32–39, 2020. USHACH, I.; ZLOTNIK, A. Biological role of granulocyte macrophage colony- stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) on cells of the myeloid lineage. Journal of Leukocyte Biology, v. 100, n. 3, p. 481– 489, 2016. VASANTHAPRASAD, V. et al. Systematic literature review and meta-analysis of the prevalence of secondary progressive multiple sclerosis in the USA, Europe, Canada, Australia, and Brazil. BMC Neurology, v. 22, n. 1, p. 1–20, 2022. WANG, Y. et al. Pathogenic TNF-α drives peripheral nerve inflammation in an Aire- deficient model of autoimmunity. Proceedings of the National Academy of Sciences of the United States of America, v. 119, n. 4, p. 1–13, 2022. 51 YANWU, Y. et al. Mesenchymal stem cells in experimental autoimmune encephalomyelitis model of multiple sclerosis: A systematic review and meta- analysis. Multiple Sclerosis and Related Disorders, v. 44, n. May, p. 102200, 2020. ZEIRA, O. et al. Adult autologous mesenchymal stem cells for the treatment of suspected non-infectious inflammatory diseases of the canine central nervous system: Safety, feasibility and preliminary clinical findings. Journal of Neuroinflammation, v. 12, n. 1, p. 1–11, 2015. ZHOU, T. et al. Challenges and advances in clinical applications of mesenchymal stromal cells. Journal of Hematology and Oncology, v. 14, n. 1, p. 1–24, 2021. 88 Declarations Ethical approval and consent to participate All experimental procedures involving animals were approved by the Ethics Committee of the Faculty of Veterinary Medicine and Zootechnics, UNESP-Botucatu (protocol No. 0171/2021). Blood collection and CSF of dogs naturally affected by MUO treated at Veterinary Neurology and collection of canine adipose tissue were performed according to the free and informed consent of the guardians of the animals. Availability of data and materials All data generated or analyzed during this study are included in this published article and its information files complementary. Competitive interests "The authors declare that they have no conflicting interests" in this section. Financing Author Contributions References 1. Cornelis I, Volk HA, De Decker S. Clinical presentation, diagnostic findings and long-term survival in large breed dogs with meningoencephalitis of unknown aetiology. Vet Rec. 2016;179(6). 2. Coates JR, Jeffery ND. Perspectives on meningoencephalomyelitis of unknown origin. Vet Clin North Am - Small Anim Pract. 2014;44(6):1157–85. 3. Dendrou CA, Fugger L, Friese MA. Immunopathology of multiple sclerosis. Nat Rev Immunol [Internet]. 2015;15(9):545–58. Available from: http://dx.doi.org/10.1038/nri3871 4. McGinley MP, Goldschmidt CH R-GA. Diagnosis and Treatment of Multiple Sclerosis: A Review. J Am Med Assoc [Internet]. 2021;325((8):765–779. 89 Available from: https://jamanetwork.com/journals/jama/article- abstract/2776694 5. Gonçalves R, De Decker S, Walmsley G, Butterfield S, Maddox TW. Inflammatory Disease Affecting the Central Nervous System in Dogs: A Retrospective Study in England (2010–2019). Front Vet Sci. 2022;8(January):1–11. 6. Constantinescu CS, Farooqi N, O’Brien K, Gran B. Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br J Pharmacol. 2011;164(4):1079–106. 7. Greer KA, Wong AK, Liu H, Famula TR, Pedersen NC, Ruhe A, et al. Necrotizing meningoencephalitis of Pug Dogs associates with dog leukocyte antigen class II and resembles acute variant forms of multiple sclerosis. Tissue Antigens. 2010;76(2):110–8. 8. Genc B, Bozan HR, Sermin Genc, Genc and K. Stem cell therapy for multiple sclerosis. Cochrane Database Syst Rev. 2019;2019(9). 9. Ravanidis S, Bogie JFJ, Donders R, Craeye D, Mays RW, Deans R, et al. Neuroinflammatory signals enhance the immunomodulatory and neuroprotective properties of multipotent adult progenitor cells. Stem Cell Res Ther [Internet]. 2015;6(1):1–17. Available from: http://dx.doi.org/10.1186/s13287-015-0169-z 10. Lee BC, Kang KS. Functional enhancement strategies for immunomodulation of mesenchymal stem cells and their therapeutic application. Stem Cell Res Ther. 2020;11(1):1–10. 11. Li M, Jiang Y, Hou Q, Zhao Y, Zhong L, Fu X. Potential pre-activation strategies for improving therapeutic efficacy of mesenchymal stem cells: current status and future prospects. Stem Cell Res Ther [Internet]. 2022;13(1):1–21. Available from: https://doi.org/10.1186/s13287-022-02822-2 12. Noronha NDC, Mizukami A, Caliári-Oliveira C, Cominal JG, Rocha JLM, 90 Covas DT, et al. Correction to: Priming approaches to improve the efficacy of mesenchymal stromal cell-based therapies (Stem Cell Research and Therapy (2019) 10 (131) DOI: 10.1186/s13287-019-1224-y). Stem Cell Res Ther. 2019;10(1):1–21. 13. Lee J, Kwon SJ, Kim JH, Jang H, Lee NK, Hwang JW, et al. Cerebrospinal fluid from Alzheimer’s disease patients as an optimal formulation for therapeutic application of mesenchymal stem cells in Alzheimer’s disease. Sci Rep [Internet]. 2019;9(1):1–9. Available from: http://dx.doi.org/10.1038/s41598-018-37252-9 14. Leijs MJC, van Buul GM, Lubberts E, Bos PK, Verhaar JAN, Hoogduijn MJ, et al. Effect of arthritic synovial fluids on the expression of immunomodulatory factors by mesenchymal stem cells: An explorative in vitro study. Front Immunol. 2012;3(AUG):1–7. 15. Farivar S, Mohamadzade Z, Shiari R, Fahimzad A. Neural differentiation of human umbilical cord mesenchymal stem cells by cerebrospinal fluid. Iran J Child Neurol. 2015;9(1):87–93. 16. Talarico LR, Schatzberg SJ. Idiopathic granulomatous and necrotising inflammatory disorders of the canine central nervous system: A review and future perspectives. J Small Anim Pract. 2010;51(3):138–49. 17. Amorim RM, Clark KC, Walker NJ, Kumar P, Herout K, Borjesson DL, et al. Placenta-derived multipotent mesenchymal stromal cells: A promising potential cell-based therapy for canine inflammatory brain disease. Stem Cell Res Ther. 2020;11(1):1–12. 18. Chung DJ, Hayashi K, Toupadakis CA, Wong A, Yellowley CE. Osteogenic proliferation and differentiation of canine bone marrow and adipose tissue derived mesenchymal stromal cells and the influence of hypoxia. Res Vet Sci [Internet]. 2012;92(1):66–75. Available from: http://dx.doi.org/10.1016/j.rvsc.2010.10.012 19. Russell KA, Chow NHC, Dukoff D, Gibson TWG, La Marre J, Betts DH, et al. 91 Characterization and immunomodulatory effects of canine adipose tissue- and bone marrow-derived mesenchymal stromal cells. PLoS One. 2016;11(12). 20. Vieira NM, Brandalise V, Zucconi E, Secco M, Strauss BE, Zatz M. Isolation, characterization, and differentiation potential of canine adipose-derived stem cells. Cell Transplant. 2010;19(3):279–89. 21. Luna ACL, Madeira MEP, Conceição TO, Moreira JALC, Laiso RAN, Maria DA. Characterization of adipose-derived stem cells of anatomical region from mice. BMC Res Notes. 2014;7(1):1–12. 22. Zhan XS, El-Ashram S, Luo DZ, Luo HN, Wang BY, Chen SF, et al. A comparative study of biological characteristics and transcriptome profiles of mesenchymal stem cells from different canine tissues. Int J Mol Sci. 2019;20(6). 23. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy [Internet]. 2006;8(4):315–7. Available from: http://dx.doi.org/10.1080/14653240600855905 24. Screven R, Kenyon E, Myers MJ, Yancy HF, Skasko M, Boxer L, et al. Immunophenotype and gene expression profile of mesenchymal stem cells derived from canine adipose tissue and bone marrow. Vet Immunol Immunopathol [Internet]. 2014;161(1–2):21–31. Available from: http://dx.doi.org/10.1016/j.vetimm.2014.06.002 25. Suzumura A. Neuron-microglia interactions in neuroinflammation. Clin Exp Neuroimmunol. 2015;6(3):225–31. 26. Yang HM, Song WJ, Li Q, Kim SY, Kim HJ, Ryu MO, et al. Canine mesenchymal stem cells treated with TNF-α and IFN-γ enhance anti- inflammatory effects through the COX-2/PGE 2 pathway. Res Vet Sci. 2018;119(May):19–26. 92 27. Kim DS, Jang IK, Lee MW, Ko YJ, Lee DH, Lee JW, et al. Enhanced Immunosuppressive Properties of Human Mesenchymal Stem Cells Primed by Interferon-γ. EBioMedicine [Internet]. 2018;28:261–73. Available from: https://doi.org/10.1016/j.ebiom.2018.01.002 28. Rodríguez-Sánchez DN, Pinto GBA, Cartarozzi LP, de Oliveira ALR, Bovolato ALC, de Carvalho M, et al. 3D-printed nerve guidance conduits multi- functionalized with canine multipotent mesenchymal stromal cells promote neuroregeneration after sciatic nerve injury in rats. Stem Cell Res Ther. 2021;12(1):1–20. 29. Ha H, Debnath B, Neamati N. Role of the CXCL8-CXCR1/2 axis in cancer and inflammatory diseases. Theranostics. 2017;7(6):1543–88. 30. Hou Y, Ryu CH, Jun JA, Kim SM, Jeong CH, Jeun SS. IL-8 enhances the angiogenic potential of human bone marrow mesenchymal stem cells by increasing vascular endothelial growth factor. Cell Biol Int. 2014;38(9):1050– 9. 31. Wang Y, Guo L, Yin X, McCarthy EC, Cheng MI, Hoang AT, et al. Pathogenic TNF-α drives peripheral nerve inflammation in an Aire-deficient model of autoimmunity. Proc Natl Acad Sci U S A. 2022;119(4):1–13. 32. Tan PH, Ji J, Yeh CC, Ji RR. Interferons in Pain and Infections: Emerging Roles in Neuro-Immune and Neuro-Glial Interactions. Front Immunol. 2021;12(November):1–14. 33. Jiang W, Xu J. Immune modulation by mesenchymal stem cells. Cell Prolif. 2020;53(1):1–16. 34. Benkhoucha M, Santiago-Raber ML, Schneiter G, Chofflon M, Funakoshi H, Nakamura T, et al. Hepatocyte growth factor inhibits CNS autoimmunity by inducing tolerogenic dendritic cells and CD25+Foxp3+ regulatory T cells. Proc Natl Acad Sci U S A. 2010;107(14):6424–9. 35. Aldhshan MS, Jhanji G, Poritsanos NJ, Mizuno TM. Glucose Stimulates Glial 93 Cell Line-Derived Neurotrophic Factor Gene Expression in Microglia through a GLUT5-Independent Mechanism. Int J Mol Sci. 2022;23(13):1–13. 36. Lima Giacobbo B, Doorduin J, Klein HC, Dierckx RAJO, Bromberg E, de Vries EFJ. Brain-Derived Neurotrophic Factor in Brain Disorders: Focus on Neuroinflammation. Mol Neurobiol. 2019;56(5):3295–312. 37. Krull AA, Setter DO, Gendron TF, Hrstka SCL, Polzin MJ, Hart J, et al. Alterations of mesenchymal stromal cells in cerebrospinal fluid: insights from transcriptomics and an ALS clinical trial. Stem Cell Res Ther. 2021;12(1):1– 14. 38. Ge W, Ren C, Duan X, Geng D, Zhang C, Liu X, et al. Differentiation of Mesenchymal Stem Cells into Neural Stem Cells Using Cerebrospinal Fluid. Cell Biochem Biophys. 2015;71(1):449–55. 39. Silva-Carvalho AÉ, Rodrigues LP, Schiavinato JL, Alborghetti MR, Bettarello G, Simões BP, et al. GVHD-derived plasma as a priming strategy of mesenchymal stem cells. Stem Cell Res Ther. 2020;11(1):1–12. 40. Saraiva M, O’Garra A. The regulation of IL-10 production by immune cells. Nat Rev Immunol [Internet]. 2010;10(3):170–81. Available from: http://dx.doi.org/10.1038/nri2711 41. Singh S, Anshita D, Ravichandiran V. MCP-1: Function, regulation, and involvement in disease. Int Immunopharmacol [Internet]. 2021;101:107598. Available from: https://doi.org/10.1016/j.intimp.2021.107598 42. Ushach I, Zlotnik A. Biological role of granulocyte macrophage colony- stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M- CSF) on cells of the myeloid lineage. J Leukoc Biol. 2016;100(3):481–9. 43. Abbas AK, Trotta E, Simeonov DR, Marson A, Bluestone JA. Revisiting IL-2: Biology and therapeutic prospects. Sci Immunol. 2018;3(25). 95 3 CONCLUSÃO GERAL O priming celular é uma técnica nova que tem por objetivo melhorar a capacidade imunomoduladora das MSCs, e o uso de fluidos biológicos é algo inédito demonstrando a importância deste estudo ser o primeiro estudo com uso de LCR e soro sanguíneo em Ad-MSCs na espécie canina. Embora o LCR e o soro de cães com MUO não tenham produzido o efeito imunomodulador significativo nas Ad- MSCs novos estudos com concentrações e tempo de estimulação diferentes são fundamentais para melhor conhecimento do perfil dessas células submetidas a um ambiente com LCR e soro de cães com MUO. A técnica priming das cAd-MSCs com o uso de INF-Ƴ+TNF-α se mostrou com um perfil com maior endência imunomodulatória e imunossupressora em comparação com o grupo controle e entre os grupos. A viabilidade das cAd-MSCs não sofreu alteração nos grupos avaliados após o priming e sua taxa metabólica não foi prejudicada quando submetidas a diferentes concentrações de LCR e Soro. O modelo experimental proposto é de suma importância para o estudo in vitro do comportamento das cAd-MSCs em ambientes que mimetizam as doenças neuroimunes como a esclerose múltipla, contudo apresentando limitações como a difuculdade de padronização do LCR e soro inflamatório. Como perspectivas futuras novos estudos com deferentes concentrações de LCR e soro podem se demonstrar resultados mais favoráveis a essas técnicas priming.