UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE MEDICINA VETERINÁRIA CAMPUS DE ARAÇATUBA MATHEUS FUJIMURA SOARES MIR-150 REGULA A CARGA PARASITÁRIA DE LEISHMANIA INFANTUM E OS NÍVEIS DE GZMB NAS PBMCS DE CÃES COM LEISHMANIOSE VISCERAL CANINA Araçatuba 2022 2 MATHEUS FUJIMURA SOARES MIR-150 REGULA A CARGA PARASITÁRIA DE LEISHMANIA INFANTUM E OS NÍVEIS DE GZMB NAS PBMCS DE CÃES COM LEISHMANIOSE VISCERAL CANINA Dissertação apresentada à Faculdade de Medicina Veterinária de Araçatuba da Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP, como parte dos requisitos para a obtenção do título de Mestre em Ciência Animal (Área de Medicina Veterinária Preventiva e Produção Animal). Orientadora: Prof.ª Associada Valéria Marçal Felix de Lima Araçatuba 2022 3 4 5 6 AGRADECIMENTOS Primeiramente quero agradecer a Deus, meu Pai, meu Amigo. Tudo é por Ele e para Ele! Aqui me faltariam palavras! Aos meus pais José Roberto e Marisa, e minha irmã Giovanna, pelo suporte durante todo o tempo de mestrado. Obrigado por entenderem as inúmeras recusas durante esse tempo e serem pacientes comigo. À Gabriele, minha linda, que durante esse tempo entendeu que eu precisaria estar dedicando muito tempo no mestrado. Obrigado por sempre me incentivar e alegrar nesse tempo! À todos meus amigos que, de alguma forma, me deram maior clareza durante esse tempo. Cito o TJ, o Wini, o Twister, o Athos, o Leli, o Gu e o Luiz. Obrigado por me esticarem tanto! Aos amigos que sempre me ajudaram a relaxar quando estive muito "pensativo". Cito aqui o Thales, o Calebe, o Dani, o Du, o Léo, o Breno e a Camila. Obrigado por me ajudarem a descontrair quando as coisas estavam tensas! À professora Valéria, que me acolheu desde o estágio de graduação. Passamos juntos pela iniciação científica, monitoria, PAADES e o mestrado! Muito obrigado por acreditar em mim e me dar oportunidades de aprender e conhecer mais a Ciência. Formamos uma excelente equipe! A todos os amigos que conquistei pela Ciência! Cito aqui meus companheiros de laboratório Sidnei, Jéssica, Jaqueline Bizi, Marilene e Gabi Venturin e todos os estagiários e ICs. Além disso, agradeço a Gabi Rebech, Jaque Poleto, Larissa Melo, Gabriane Porcino, Camila, Débora, Amanda, Yuri e Thiago Vidotto (seu conteúdo mudou minha visão da Ciência!!). À minha banca de defesa, Dra. Flávia Lombardi e Dra. Sandra Muxel, por estarem sempre atualizadas e antenadas às novidades do mundo das Leishmanias e do ncRNAs! Obrigado por sempre estarem dispostas a ensinar e a corrigir. A Academia é melhor por causa de vocês! À CAPES e à FAPESP (processo 2020/00565-1) pelo financiamento do projeto! javascript:openProcess('316334',%20'false') 7 “Bem sei que tudo podes, e nenhum dos teus planos pode ser frustrado." Jó 42:2 https://www.bibliaonline.com.br/ara/j%C3%B3/42/2+ 8 SOARES, M.F. et al. miR-150 regula a carga parasitária de Leishmania infantum e os níveis de GZMB nas PBMCs de cães com Leishmaniose Visceral Canina. 2022. 100 f. Dissertação (Mestrado) - Faculdade de Medicina Veterinária, Universidade Estadual Paulista, Araçatuba, 2022. RESUMO A Leishmania infantum causa a leishmaniose visceral, uma doença tropical negligenciada que pode modular a resposta imune do hospedeiro por meio de pequenos RNAs não codificantes chamados microRNAs (miRNAs). Alguns miRNAs são expressos diferencialmente em células mononucleares do sangue periférico (PBMCs) de cães com leishmaniose visceral canina (LCan), incluindo o miR-150, cuja expressão está diminuída. Embora o miR-150 esteja negativamente correlacionado com a carga parasitária de L. infantum, não está claro se o miR-150 afeta diretamente a carga parasitária de L. infantum e (em caso afirmativo) como esse miRNA contribuiria para a infecção. Aqui, isolamos as PBMCs de 14 cães naturalmente infectados (grupo LCan) e seis cães saudáveis (grupo controle) e as tratamos in vitro com o mimetizador ou o inibidor do miR-150. Medimos a carga parasitária de L. infantum usando o qPCR e comparamos os tratamentos. Também medimos os níveis de proteína-alvo do miR-150 preditas in silico (STAT1, TNF-α, HDAC8 e GZMB) usando citometria de fluxo ou ensaios imunoenzimáticos. O mimetizador do miR-150 diminuiu a carga parasitária de L. infantum nas PBMCs de cães do grupo LCan. Também descobrimos que a inibição do miR-150 reduziu os níveis de GZMB. Esses achados demosntram que o miR-150 exerce uma função importante na infecção por L. infantum nas PBMCs caninas, o que poderia ser usado para mais estudos visando o desenvolvimento de drogas. Palavras-chave: LCan. Leishmania infantum. miRNA. miR-150. GZMB 9 SOARES, M.F. et al. miR-150 regulates the Leishmania infantum parasitic load and GZMB levels in peripheral blood mononuclear cells of dogs with canine visceral leishmaniasis. 2022. 100 f. Dissertação (Mestrado) - Faculdade de Medicina Veterinária, Universidade Estadual Paulista, Araçatuba, 2022. ABSTRACT Leishmania infantum causes visceral leishmaniasis, a neglected tropical disease that can modulate the host immune response by altering the expression of small non-coding RNAs called microRNAs (miRNAs). Some miRNAs are differentially expressed in peripheral blood mononuclear cells (PBMCs) of dogs with visceral canine leishmaniasis (CanL), like the down-regulated miR-150. Even though miR-150 is negatively correlated with L. infantum parasitic load, it is unclear if miR-150 directly affects L. infantum parasitic load and (if so) how this miRNA would contribute to infection. Here, we isolated PBMCs from 14 naturally infected dogs (CanL group) and six healthy dogs (Control group) and treated them in vitro with miR-150 mimic or inhibitor. We measured L. infantum parasitic load using qPCR and compared treatments. We also measured miR-150 in silico predicted target protein levels (STAT1, TNF-α, HDAC8, and GZMB) using flow cytometry or enzyme-linked immunosorbent assays. Increasing miR-150 activity diminished L. infantum parasitic load in CanL PBMCs. We also found that inhibition of miR- 150 reduced GZMB levels. These findings demonstrate that miR-150 plays an important role in L. infantum infection in canine PBMCs, and these findings merit further studies aiming at drug development. Key-words: CanL. Leishmania infantum. miRNA. miR-150. GZMB. 64 APÊNDICE - REFERÊNCIAS DA INTRODUÇÃO GERAL 1. Steverding D. The history of leishmaniasis. Parasit Vectors. 2017;10: 82. doi:10.1186/s13071-017-2028-5 2. Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. Leishmaniasis Worldwide and Global Estimates of Its Incidence. Kirk M, editor. PLoS One. 2012;7: e35671. doi:10.1371/journal.pone.0035671 3. de Freitas EO, Leoratti FM de S, Freire-de-Lima CG, Morrot A, Feijó DF. The Contribution of Immune Evasive Mechanisms to Parasite Persistence in Visceral Leishmaniasis. Front Immunol. 2016;7: 153. doi:10.3389/fimmu.2016.00153 4. Maroli M, Feliciangeli MD, Bichaud L, Charrel RN, Gradoni L. Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern. Med Vet Entomol. 2013;27: 123–147. doi:10.1111/j.1365-2915.2012.01034.x 5. Cecílio P, Cordeiro-da-Silva A, Oliveira F. Sand flies: Basic information on the vectors of leishmaniasis and their interactions with Leishmania parasites. Commun Biol. 2022;5: 305. doi:10.1038/s42003-022-03240-z 6. Ribeiro RR, Michalick MSM, da Silva ME, dos Santos CCP, Frézard FJG, da Silva SM. Canine Leishmaniasis: An Overview of the Current Status and Strategies for Control. Biomed Res Int. 2018;2018: 1–12. doi:10.1155/2018/3296893 7. Esch KJ, Petersen CA. Transmission and Epidemiology of Zoonotic Protozoal Diseases of Companion Animals. Clin Microbiol Rev. 2013;26: 58–85. doi:10.1128/CMR.00067-12 65 8. Bates PA. Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies. Int J Parasitol. 2007;37: 1097–1106. doi:10.1016/j.ijpara.2007.04.003 9. Kelly PH, Bahr SM, Serafim TD, Ajami NJ, Petrosino JF, Meneses C, et al. The Gut Microbiome of the Vector Lutzomyia longipalpis Is Essential for Survival of Leishmania infantum. Beverley SM, Sher A, editors. MBio. 2017;8. doi:10.1128/mBio.01121-16 10. Pearson RD, Sousa A d. Q. Clinical Spectrum of Leishmaniasis. Clin Infect Dis. 1996;22: 1–13. doi:10.1093/clinids/22.1.1 11. Herricks JR, Hotez PJ, Wanga V, Coffeng LE, Haagsma JA, Basáñez M- G, et al. The global burden of disease study 2013: What does it mean for the NTDs? Zhou X-N, editor. PLoS Negl Trop Dis. 2017;11: e0005424. doi:10.1371/journal.pntd.0005424 12. Álvarez-Hernández D-A, Rivero-Zambrano L, Martínez-Juárez L-A, García-Rodríguez-Arana R. Overcoming the global burden of neglected tropical diseases. Ther Adv Infect Dis. 2020;7: 204993612096644. doi:10.1177/2049936120966449 13. Costa DNCC, Bermudi PMMB, Rodas LAC, Nunes CM, Hiramoto RM, Tolezano JE, et al. Human visceral leishmaniasis and relationship with vector and canine control measures. Rev Saude Publica. 2018;52: 92. doi:10.11606/S1518-8787.2018052000381 14. Nicolle C, Comte C. Origine canine du kala-azar. Bull Soc Pathol Exot. 1908;1: 299–301. 15. Araújo VEM de, Pinheiro LC, Almeida MC de M, Menezes FC de, Morais MHF, Reis IA, et al. Relative Risk of Visceral Leishmaniasis in Brazil: A Spatial Analysis in Urban Area. Kamhawi S, editor. PLoS Negl Trop Dis. 2013;7: e2540. doi:10.1371/journal.pntd.0002540 66 16. Rangel EF, Vilela ML. Lutzomyia longipalpis (Diptera, Psychodidae, Phlebotominae) and urbanization of visceral leishmaniasis in Brazil. Cad Saude Publica. 2008;24: 2948–2952. doi:10.1590/S0102- 311X2008001200025 17. Marcondes M, Day MJ. Current status and management of canine leishmaniasis in Latin America. Res Vet Sci. 2019;123: 261–272. doi:10.1016/j.rvsc.2019.01.022 18. Solano-Gallego L, Koutinas A, Miró G, Cardoso L, Pennisi MG, Ferrer L, et al. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Vet Parasitol. 2009;165: 1–18. doi:10.1016/j.vetpar.2009.05.022 19. Silva FL, Oliveira RG, Silva TMA, Xavier MN, Nascimento EF, Santos RL. Venereal transmission of canine visceral leishmaniasis. Vet Parasitol. 2009;160: 55–59. doi:10.1016/j.vetpar.2008.10.079 20. da Silva SM, Ribeiro VM, Ribeiro RR, Tafuri WL, Melo MN, Michalick MSM. First report of vertical transmission of Leishmania (Leishmania) infantum in a naturally infected bitch from Brazil. Vet Parasitol. 2009;166: 159–162. doi:10.1016/j.vetpar.2009.08.011 21. de Freitas E, Melo MN, da Costa-Val AP, Michalick MSM. Transmission of Leishmania infantum via blood transfusion in dogs: Potential for infection and importance of clinical factors. Vet Parasitol. 2006;137: 159–167. doi:10.1016/j.vetpar.2005.12.011 22. Coutinho MTZ, Linardi PM. Can fleas from dogs infected with canine visceral leishmaniasis transfer the infection to other mammals? Vet Parasitol. 2007;147: 320–325. doi:10.1016/j.vetpar.2007.04.008 67 23. Ferreira MGPA, Fattori KR, Souza F, Lima VMF. Potential role for dog fleas in the cycle of Leishmania spp. Vet Parasitol. 2009;165: 150–154. doi:10.1016/j.vetpar.2009.06.026 24. de Morais RCS, Gonçalves S da C, Costa PL, da Silva KG, da Silva FJ, Silva RPE, et al. Detection of Leishmania infantum in animals and their ectoparasites by conventional PCR and real time PCR. Exp Appl Acarol. 2013;59: 473–481. doi:10.1007/s10493-012-9611-4 25. Coutinho MTZ, Bueno LL, Sterzik A, Fujiwara RT, Botelho JR, De Maria M, et al. Participation of Rhipicephalus sanguineus (Acari: Ixodidae) in the epidemiology of canine visceral leishmaniasis. Vet Parasitol. 2005;128: 149–155. doi:10.1016/j.vetpar.2004.11.011 26. Alvar J, Cañavate C, Molina R, Moreno J, Nieto J. Canine Leishmaniasis. Advances in Parasitology. 2004. pp. 1–88. doi:10.1016/S0065- 308X(04)57001-X 27. Brandonisio O, Carelli G, Ceci L, Consenti B, Fasanella A, Puccini V. Canine leishmaniasis in the Gargano promontory (Apulia, South Italy). Eur J Epidemiol. 1992;8: 273–276. doi:10.1007/BF00144813 28. Barati M, Mohebali M, Alimohammadian MH, Khamesipour A, Akhoundi B, Zarei Z. Canine visceral leishmaniasis: seroprevalence survey of asymptomatic dogs in an endemic area of northwestern Iran. J Parasit Dis Off Organ Indian Soc Parasitol. 2015;39: 221. doi:10.1007/S12639-013- 0325-2 29. Reis AB, Teixeira-Carvalho A, Giunchetti RC, Guerra LL, Carvalho MG, Mayrink W, et al. Phenotypic features of circulating leucocytes as immunological markers for clinical status and bone marrow parasite density in dogs naturally infected by Leishmania chagasi. Clin Exp Immunol. 2006;146: 303–311. doi:10.1111/j.1365-2249.2006.03206.x 68 30. Coura-Vital W, Marques MJ, Giunchetti RC, Teixeira-Carvalho A, Moreira ND, Vitoriano-Souza J, et al. Humoral and cellular immune responses in dogs with inapparent natural Leishmania infantum infection. Vet J. 2011;190: e43–e47. doi:10.1016/j.tvjl.2011.04.005 31. Laurenti MD, Rossi CN, Matta VLR da, Tomokane TY, Corbett CEP, Secundino NFC, et al. Asymptomatic dogs are highly competent to transmit Leishmania (Leishmania) infantum chagasi to the natural vector. Vet Parasitol. 2013;196: 296–300. doi:10.1016/J.VETPAR.2013.03.017 32. Koutinas A, Polizopoulou Z, Saridomichelakis M, Argyriadis D, Fytianou A, Plevraki K. Clinical considerations on canine visceral leishmaniasis in Greece: a retrospective study of 158 cases (1989-1996). J Am Anim Hosp Assoc. 1999;35: 376–383. doi:10.5326/15473317-35-5-376 33. Baneth G, Koutinas AF, Solano-Gallego L, Bourdeau P, Ferrer L. Canine leishmaniosis – new concepts and insights on an expanding zoonosis: part one. Trends Parasitol. 2008;24: 324–330. doi:10.1016/j.pt.2008.04.001 34. Nicolato RDC, Abreu RT de, Roatt BM, Aguiar-Soares RDDO, Reis LES, Carvalho MDG, et al. Clinical Forms of Canine Visceral Leishmaniasis in Naturally Leishmania infantum–Infected Dogs and Related Myelogram and Hemogram Changes. Arez AP, editor. PLoS One. 2013;8: e82947. doi:10.1371/journal.pone.0082947 35. Ribeiro RR, Silva SM da, Fulgêncio G de O, Michalick MSM, Frézard FJG. Relationship between clinical and pathological signs and severity of canine leishmaniasis. Rev Bras Parasitol Veterinária. 2013;22: 373–378. doi:10.1590/S1984-29612013000300009 36. Maia C, Campino L. Biomarkers Associated With Leishmania infantum Exposure, Infection, and Disease in Dogs. Front Cell Infect Microbiol. 2018;8. doi:10.3389/fcimb.2018.00302 69 37. Reis AB, Martins-Filho OA, Teixeira-Carvalho A, Carvalho MG, Mayrink W, França-Silva JC, et al. Parasite density and impaired biochemical/hematological status are associated with severe clinical aspects of canine visceral leishmaniasis. Res Vet Sci. 2006;81: 68–75. doi:10.1016/j.rvsc.2005.09.011 38. Saridomichelakis MN, Mylonakis ME, Leontides LS, Koutinas AF, Billinis C, Kontos VI. Evaluation of lymph node and bone marrow cytology in the diagnosis of canine leishmaniasis (Leishmania infantum) in symptomatic and asymptomatic dogs. Am J Trop Med Hyg. 2005;73: 82–86. doi:10.4269/ajtmh.2005.73.82 39. Sundar S, Rai M. Laboratory Diagnosis of Visceral Leishmaniasis. Clin Vaccine Immunol. 2002;9: 951–958. doi:10.1128/CDLI.9.5.951-958.2002 40. Porrozzi R, Santos da Costa M V., Teva A, Falqueto A, Ferreira AL, dos Santos CD, et al. Comparative Evaluation of Enzyme-Linked Immunosorbent Assays Based on Crude and Recombinant Leishmanial Antigens for Serodiagnosis of Symptomatic and Asymptomatic Leishmania infantum Visceral Infections in Dogs. Clin Vaccine Immunol. 2007;14: 544– 548. doi:10.1128/CVI.00420-06 41. Fraga DBM, Pacheco LV, Borja LS, Tuy PG da SE, Bastos LA, Solcà M da S, et al. The Rapid Test Based on Leishmania infantum Chimeric rK28 Protein Improves the Diagnosis of Canine Visceral Leishmaniasis by Reducing the Detection of False-Positive Dogs. Picado A, editor. PLoS Negl Trop Dis. 2016;10: e0004333. doi:10.1371/journal.pntd.0004333 42. Silva RC, Richini-Pereira VB, Kikuti M, Marson PM, Langoni H. Detection of Leishmania (L.) infantum in stray dogs by molecular techniques with sensitive species-specific primers. Vet Q. 2017;37: 23–30. doi:10.1080/01652176.2016.1252073 70 43. Travi BL, Cordeiro-da-Silva A, Dantas-Torres F, Miró G. Canine visceral leishmaniasis: Diagnosis and management of the reservoir living among us. Büscher P, editor. PLoS Negl Trop Dis. 2018;12: e0006082. doi:10.1371/journal.pntd.0006082 44. Coura-Vital W, Ker HG, Roatt BM, Aguiar-Soares RDO, Leal GGDA, Moreira NDD, et al. Evaluation of Change in Canine Diagnosis Protocol Adopted by the Visceral Leishmaniasis Control Program in Brazil and a New Proposal for Diagnosis. Zamboni DS, editor. PLoS One. 2014;9: e91009. doi:10.1371/journal.pone.0091009 45. Dantas-Torres F, Miró G, Baneth G, Bourdeau P, Breitschwerdt E, Capelli G, et al. Canine Leishmaniasis Control in the Context of One Health. Emerg Infect Dis. 2019;25: 1–4. doi:10.3201/eid2512.190164 46. Pereira M, Valério-Bolas A, Santos-Mateus D, Alexandre-Pires G, Santos M, Rodrigues A, et al. Canine neutrophils activate effector mechanisms in response to Leishmania infantum. Vet Parasitol. 2017;248: 10–20. doi:10.1016/j.vetpar.2017.10.008 47. Ribeiro-Gomes FL, Sacks D. The influence of early neutrophil-Leishmania interactions on the host immune response to infection. Front Cell Infect Microbiol. 2012;2: 59. doi:10.3389/fcimb.2012.00059 48. Toepp AJ, Petersen CA. The balancing act: Immunology of leishmaniosis. Res Vet Sci. 2020;130: 19–25. doi:10.1016/j.rvsc.2020.02.004 49. Regli IB, Passelli K, Hurrell BP, Tacchini-Cottier F. Survival Mechanisms Used by Some Leishmania Species to Escape Neutrophil Killing. Front Immunol. 2017;8: 16. doi:10.3389/fimmu.2017.01558 50. Montserrat-Sangrà S, Alborch L, Ordeix L, Solano-Gallego L. TLR-2 and TLR-4 transcriptions in unstimulated blood from dogs with leishmaniosis due to Leishmania infantum at the time of diagnosis and during follow-up 71 treatment. Vet Parasitol. 2016;228: 172–179. doi:10.1016/j.vetpar.2016.09.005 51. Netea MG, Van der Graaf C, Van der Meer JWM, Kullberg BJ. Toll-like receptors and the host defense against microbial pathogens: bringing specificity to the innate-immune system. J Leukoc Biol. 2004;75: 749–755. doi:10.1189/jlb.1103543 52. Van Assche T, Deschacht M, da Luz RAI, Maes L, Cos P. Leishmania– macrophage interactions: Insights into the redox biology. Free Radic Biol Med. 2011;51: 337–351. doi:10.1016/j.freeradbiomed.2011.05.011 53. Hosein S, Blake DP, Solano-Gallego L. Insights on adaptive and innate immunity in canine leishmaniosis. Parasitology. 2017;144: 95–115. doi:10.1017/S003118201600055X 54. Shio MT, Hassani K, Isnard A, Ralph B, Contreras I, Gomez MA, et al. Host Cell Signalling and Leishmania Mechanisms of Evasion. J Trop Med. 2012;2012: 1–14. doi:10.1155/2012/819512 55. Zamboni DS, Sacks DL. Inflammasomes and Leishmania: in good times or bad, in sickness or in health. Curr Opin Microbiol. 2019;52: 70–76. doi:10.1016/j.mib.2019.05.005 56. Lima-Junior DS, Costa DL, Carregaro V, Cunha LD, Silva ALN, Mineo TWP, et al. Inflammasome-derived IL-1β production induces nitric oxide– mediated resistance to Leishmania. Nat Med. 2013;19: 909–915. doi:10.1038/nm.3221 57. de Carvalho RVH, Andrade WA, Lima-Junior DS, Dilucca M, de Oliveira C V., Wang K, et al. Leishmania Lipophosphoglycan Triggers Caspase-11 and the Non-canonical Activation of the NLRP3 Inflammasome. Cell Rep. 2019;26: 429-437.e5. doi:10.1016/j.celrep.2018.12.047 72 58. Lefèvre L, Lugo-Villarino G, Meunier E, Valentin A, Olagnier D, Authier H, et al. The C-type Lectin Receptors Dectin-1, MR, and SIGNR3 Contribute Both Positively and Negatively to the Macrophage Response to Leishmania infantum. Immunity. 2013;38: 1038–1049. doi:10.1016/j.immuni.2013.04.010 59. Sacks D, Sher A. Evasion of innate immunity by parasitic protozoa. Nat Immunol. 2002;3: 1041–1047. doi:10.1038/ni1102-1041 60. Ueno N, Bratt CL, Rodriguez NE, Wilson ME. Differences in human macrophage receptor usage, lysosomal fusion kinetics and survival between logarithmic and metacyclic Leishmania infantum chagasi promastigotes. Cell Microbiol. 2009;11: 1827–1841. doi:10.1111/j.1462- 5822.2009.01374.x 61. Rodriguez NE, Gaur U, Wilson ME. Role of caveolae in Leishmania chagasi phagocytosis and intracellular survival in macrophages. Cell Microbiol. 2006;8: 1106–1120. doi:10.1111/j.1462-5822.2006.00695.x 62. Barr SD, Gedamu L. Role of Peroxidoxins in Leishmania chagasi Survival. J Biol Chem. 2003;278: 10816–10823. doi:10.1074/jbc.M212990200 63. Plewes KA, Barr SD, Gedamu L. Iron Superoxide Dismutases Targeted to the Glycosomes of Leishmania chagasi Are Important for Survival. Infect Immun. 2003;71: 5910–5920. doi:10.1128/IAI.71.10.5910-5920.2003 64. Longoni SS, Sánchez-Moreno M, López JER, Marín C. Leishmania infantum secreted iron superoxide dismutase purification and its application to the diagnosis of canine Leishmaniasis. Comp Immunol Microbiol Infect Dis. 2013;36: 499–506. doi:10.1016/j.cimid.2013.05.004 65. Gupta AK, Ghosh K, Palit S, Barua J, Das PK, Ukil A. Leishmania donovani inhibits inflammasome-dependent macrophage activation by 73 exploiting the negative regulatory proteins A20 and UCP2. FASEB J. 2017;31: 5087–5101. doi:10.1096/fj.201700407R 66. Shio MT, Christian JG, Jung JY, Chang K-P, Olivier M. PKC/ROS- Mediated NLRP3 Inflammasome Activation Is Attenuated by Leishmania Zinc-Metalloprotease during Infection. Burleigh BA, editor. PLoS Negl Trop Dis. 2015;9: e0003868. doi:10.1371/journal.pntd.0003868 67. Beasley EA, Pessôa-Pereira D, Scorza BM, Petersen CA. Epidemiologic, clinical and immunological consequences of co-infections during canine leishmaniosis. Animals. 2021. p. 3206. doi:10.3390/ani11113206 68. Barbiéri CL. Immunology of canine leishmaniasis. Parasite Immunol. 2006;28: 329–337. doi:10.1111/j.1365-3024.2006.00840.x 69. Alexandre-Pires G, de Brito MTV, Algueró C, Martins C, Rodrigues OR, da Fonseca IP, et al. Canine leishmaniosis. Immunophenotypic profile of leukocytes in different compartments of symptomatic, asymptomatic and treated dogs. Vet Immunol Immunopathol. 2010;137: 275–283. doi:10.1016/j.vetimm.2010.06.007 70. Sanchez-Robert E, Altet L, Utzet-Sadurni M, Giger U, Sanchez A, Francino O. Slc11a1 (formerly Nramp1) and susceptibility to canine visceral leishmaniasis. Vet Res. 2008;39: 36. doi:10.1051/vetres:2008013 71. Altet L, Francino O, Solano-Gallego L, Renier C, Sánchez A. Mapping and sequencing of the canine NRAMP1 gene and identification of mutations in leishmaniasis-susceptible dogs. Infect Immun. 2002;70: 2763–2771. doi:10.1128/IAI.70.6.2763-2771.2002 72. Quinnell RJ, Kennedy LJ, Barnes A, Courtenay O, Dye C, Garcez LM, et al. Susceptibility to visceral leishmaniasis in the domestic dog is associated with MHC class II polymorphism. Immunogenetics. 2003;55: 23–28. doi:10.1007/s00251-003-0545-1 74 73. Solano-Gallego L, Llull J, Ramos G, Riera C, Arboix M, Alberola J, et al. The Ibizian hound presents a predominantly cellular immune response against natural Leishmania infection. Vet Parasitol. 2000;90: 37–45. doi:10.1016/S0304-4017(00)00223-5 74. Strauss-Ayali D, Baneth G, Shor S, Okano F, Jaffe CL. Interleukin-12 augments a Th1-type immune response manifested as lymphocyte proliferation and interferon gamma production in Leishmania infantum- infected dogs. Int J Parasitol. 2005;35: 63–73. doi:10.1016/j.ijpara.2004.10.015 75. Kumar R, Nylén S. Immunobiology of visceral leishmaniasis. Front Immunol. 2012;3: 251. doi:10.3389/fimmu.2012.00251 76. Song Y, Wang N, Chen L, Fang L. Tr1 Cells as a Key Regulator for Maintaining Immune Homeostasis in Transplantation. doi:10.3389/fimmu.2021.671579 77. Wherry EJ. T cell exhaustion. Nat Immunol. 2011;12: 492–499. doi:10.1038/ni.2035 78. Chiku VM, Silva KLO, de Almeida BFM, Venturin GL, Leal AAC, de Martini CC, et al. PD-1 function in apoptosis of T lymphocytes in canine visceral leishmaniasis. Immunobiology. 2016;221: 879–888. doi:10.1016/j.imbio.2016.03.007 79. Lima VMF de, Fattori KR, de Souza F, Eugênio FR, Santos PSP dos, Rozza DB, et al. Apoptosis in T lymphocytes from spleen tissue and peripheral blood of L. (L.) chagasi naturally infected dogs. Vet Parasitol. 2012;184: 147–153. doi:10.1016/j.vetpar.2011.08.024 80. Miles SA, Conrad SM, Alves RG, Jeronimo SMB, Mosser DM. A role for IgG immune complexes during infection with the intracellular pathogen Leishmania. J Exp Med. 2005;201: 747–754. doi:10.1084/jem.20041470 75 81. Esch KJ, Schaut RG, Lamb IM, Clay G, Morais Lima ÁL, do Nascimento PRP, et al. Activation of Autophagy and Nucleotide-Binding Domain Leucine-Rich Repeat–Containing-Like Receptor Family, Pyrin Domain– Containing 3 Inflammasome during Leishmania infantum–Associated Glomerulonephritis. Am J Pathol. 2015;185: 2105–2117. doi:10.1016/j.ajpath.2015.04.017 82. Cortese L, Piantedosi D, Ciaramella P, Pero ME, Sica M, Ruggiero G, et al. Secondary immune-mediated thrombocytopenia in dogs naturally infected by Leishmania infantum. Vet Rec. 2009;164: 778–782. doi:10.1136/vr.164.25.778 83. Costa FAL, Goto H, Saldanha LCB, Silva SMMS, Sinhorini IL, Silva TC, et al. Histopathologic Patterns of Nephropathy in Naturally Acquired Canine Visceral Leishmaniasis. Vet Pathol. 2003;40: 677–684. doi:10.1354/vp.40- 6-677 84. Wang R-X, Yu C-R, Dambuza IM, Mahdi RM, Dolinska MB, Sergeev Y V., et al. Interleukin-35 induces regulatory B cells that suppress autoimmune disease. Nat Med. 2014;20: 633–641. doi:10.1038/nm.3554 85. Liu Y, Chen Y, Li Z, Han Y, Sun Y, Wang Q, et al. Role of IL-10-producing regulatory B cells in control of cerebral malaria in Plasmodium berghei infected mice. Eur J Immunol. 2013;43: 2907–2918. doi:10.1002/eji.201343512 86. Siewe B, Wallace J, Rygielski S, Stapleton JT, Martin J, Deeks SG, et al. Regulatory B Cells Inhibit Cytotoxic T Lymphocyte (CTL) Activity and Elimination of Infected CD4 T Cells after In Vitro Reactivation of HIV Latent Reservoirs. Unutmaz D, editor. PLoS One. 2014;9: e92934. doi:10.1371/journal.pone.0092934 87. Schaut RG, Lamb IM, Toepp AJ, Scott B, Mendes-Aguiar CO, Coutinho JF V., et al. Regulatory IgD hi B Cells Suppress T Cell Function via IL-10 and 76 PD-L1 during Progressive Visceral Leishmaniasis. J Immunol. 2016;196: 4100–4109. doi:10.4049/jimmunol.1502678 88. Gómez-Ochoa P, Castillo JA, Gascón M, Zarate JJ, Alvarez F, Couto CG. Use of domperidone in the treatment of canine visceral leishmaniasis: A clinical trial. Vet J. 2009;179: 259–263. doi:10.1016/j.tvjl.2007.09.014 89. Sabaté D, Llinás J, Homedes J, Sust M, Ferrer L. A single-centre, open- label, controlled, randomized clinical trial to assess the preventive efficacy of a domperidone-based treatment programme against clinical canine leishmaniasis in a high prevalence area. Prev Vet Med. 2014;115: 56–63. doi:10.1016/j.prevetmed.2014.03.010 90. Rebech GT, Venturin GL, Siqueira Ito LT, Bragato JP, de Carvalho Fonseca BS, Melo LM, et al. PD-1 regulates leishmanicidal activity and IL- 17 in dogs with leishmaniasis. Vet Immunol Immunopathol. 2020;219: 109970. doi:10.1016/j.vetimm.2019.109970 91. Afrin F, Khan I, Hemeg HA. Leishmania-Host Interactions—An Epigenetic Paradigm. Front Immunol. 2019;10: 492. doi:10.3389/fimmu.2019.00492 92. Bartel DP. MicroRNAs. Cell. 2004;116: 281–297. doi:10.1016/S0092- 8674(04)00045-5 93. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391: 806–811. doi:10.1038/35888 94. Lee C-T, Risom T, Strauss WM. Evolutionary Conservation of MicroRNA Regulatory Circuits: An Examination of MicroRNA Gene Complexity and Conserved MicroRNA-Target Interactions through Metazoan Phylogeny. DNA Cell Biol. 2007;26: 209–218. doi:10.1089/dna.2006.0545 77 95. Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Maller B, et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature. 2000;408: 86–89. doi:10.1038/35040556 96. Bartel DP. MicroRNAs: Target Recognition and Regulatory Functions. Cell. 2009;136: 215–233. doi:10.1016/j.cell.2009.01.002 97. Condrat CE, Thompson DC, Barbu MG, Bugnar OL, Boboc A, Cretoiu D, et al. miRNAs as Biomarkers in Disease: Latest Findings Regarding Their Role in Diagnosis and Prognosis. Cells. 2020;9: 276. doi:10.3390/cells9020276 98. Saliminejad K, Khorram Khorshid HR, Soleymani Fard S, Ghaffari SH. An overview of microRNAs: Biology, functions, therapeutics, and analysis methods. J Cell Physiol. 2019;234: 5451–5465. doi:10.1002/jcp.27486 99. Acuña SM, Floeter-Winter LM, Muxel SM. MicroRNAs: Biological Regulators in Pathogen–Host Interactions. Cells. 2020;9: 113. doi:10.3390/cells9010113 100. Olena AF, Patton JG. Genomic organization of microRNAs. J Cell Physiol. 2009;222: n/a-n/a. doi:10.1002/jcp.21993 101. Lee Y, Kim M, Han J, Yeom K-H, Lee S, Baek SH, et al. MicroRNA genes are transcribed by RNA polymerase II. EMBO J. 2004;23: 4051–4060. doi:10.1038/sj.emboj.7600385 102. Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature. 2003;425: 415–419. doi:10.1038/nature01957 78 103. Yi R, Qin Y, Macara IG, Cullen BR. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev. 2003;17: 3011– 3016. doi:10.1101/gad.1158803 104. Ketting RF, Fischer SEJ, Bernstein E, Sijen T, Hannon GJ, Plasterk RHA. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 2001;15: 2654–2659. doi:10.1101/gad.927801 105. Kobayashi H, Tomari Y. RISC assembly: Coordination between small RNAs and Argonaute proteins. Biochim Biophys Acta - Gene Regul Mech. 2016;1859: 71–81. doi:10.1016/j.bbagrm.2015.08.007 106. Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, Nishikura K, et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature. 2005;436: 740–744. doi:10.1038/nature03868 107. O’Carroll D, Schaefer A. General Principals of miRNA Biogenesis and Regulation in the Brain. Neuropsychopharmacology. 2013;38: 39–54. doi:10.1038/npp.2012.87 108. Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell. 1993;75: 855–862. doi:10.1016/0092-8674(93)90530-4 109. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin- 4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993;75: 843–854. doi:10.1016/0092-8674(93)90529-Y 110. Olsen PH, Ambros V. The lin-4 Regulatory RNA Controls Developmental Timing in Caenorhabditis elegans by Blocking LIN-14 Protein Synthesis after the Initiation of Translation. Dev Biol. 1999;216: 671–680. doi:10.1006/dbio.1999.9523 79 111. Bartel DP. Metazoan MicroRNAs. Cell. 2018;173: 20–51. doi:10.1016/j.cell.2018.03.006 112. Kawahara Y, Zinshteyn B, Sethupathy P, Iizasa H, Hatzigeorgiou AG, Nishikura K. Redirection of Silencing Targets by Adenosine-to-Inosine Editing of miRNAs. Science (80- ). 2007;315: 1137–1140. doi:10.1126/science.1138050 113. Ameres SL, Zamore PD. Diversifying microRNA sequence and function. Nat Rev Mol Cell Biol. 2013;14: 475–488. doi:10.1038/nrm3611 114. Behm-Ansmant I, Rehwinkel J, Doerks T, Stark A, Bork P, Izaurralde E. mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev. 2006;20: 1885–1898. doi:10.1101/gad.1424106 115. Younger ST, Corey DR. Transcriptional gene silencing in mammalian cells by miRNA mimics that target gene promoters. Nucleic Acids Res. 2011;39: 5682–5691. doi:10.1093/nar/gkr155 116. Kim DH, Sætrom P, Snøve O, Rossi JJ. MicroRNA-directed transcriptional gene silencing in mammalian cells. Proc Natl Acad Sci. 2008;105: 16230– 16235. doi:10.1073/pnas.0808830105 117. Zhang Y, Fan M, Zhang X, Huang F, Wu K, Zhang J, et al. Cellular microRNAs up-regulate transcription via interaction with promoter TATA- box motifs. RNA. 2014;20: 1878–1889. doi:10.1261/rna.045633.114 118. Vasudevan S, Tong Y, Steitz JA. Switching from Repression to Activation: MicroRNAs Can Up-Regulate Translation. Science (80- ). 2007;318: 1931–1934. doi:10.1126/science.1149460 119. Makarova JA, Shkurnikov MU, Wicklein D, Lange T, Samatov TR, Turchinovich AA, et al. Intracellular and extracellular microRNA: An update 80 on localization and biological role. Prog Histochem Cytochem. 2016;51: 33–49. doi:10.1016/j.proghi.2016.06.001 120. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9: 654–659. doi:10.1038/ncb1596 121. Lin J, Li J, Huang B, Liu J, Chen X, Chen X-M, et al. Exosomes: Novel Biomarkers for Clinical Diagnosis. Sci World J. 2015;2015: 1–8. doi:10.1155/2015/657086 122. Hata A, Lieberman J. Dysregulation of microRNA biogenesis and gene silencing in cancer. Sci Signal. 2015;8: re3. doi:10.1126/scisignal.2005825 123. Ushio N, Rahman M, Maemura T, Lai Y, Iwanaga T, Kawaguchi H, et al. Identification of dysregulated microRNAs in canine malignant melanoma. Oncol Lett. 2018;17: 1080. doi:10.3892/ol.2018.9692 124. Foiani G, Guelfi G, Mandara MT. MicroRNA Dysregulation in Canine Meningioma: RT-qPCR Analysis of Formalin-Fixed Paraffin-Embedded Samples. J Neuropathol Exp Neurol. 2021;80: 769–775. doi:10.1093/jnen/nlab057 125. Hussen BM, Hidayat HJ, Salihi A, Sabir DK, Taheri M, Ghafouri-Fard S. MicroRNA: A signature for cancer progression. Biomed Pharmacother. 2021;138: 111528. doi:10.1016/j.biopha.2021.111528 126. Lemaire J, Mkannez G, Guerfali FZ, Gustin C, Attia H, Sghaier RM, et al. MicroRNA Expression Profile in Human Macrophages in Response to Leishmania major Infection. Valenzuela JG, editor. PLoS Negl Trop Dis. 2013;7: e2478. doi:10.1371/journal.pntd.0002478 81 127. Geraci NS, Tan JC, Mcdowell MA. Characterization of microRNA expression profiles in Leishmania-infected human phagocytes. Parasite Immunol. 2015;37: 43–51. doi:10.1111/pim.12156 128. Tiwari N, Kumar V, Gedda MR, Singh AK, Singh VK, Singh SP, et al. Identification and Characterization of miRNAs in Response to Leishmania donovani Infection: Delineation of Their Roles in Macrophage Dysfunction. Front Microbiol. 2017;8: 314. doi:10.3389/fmicb.2017.00314 129. Soares MF, Melo LM, Bragato JP, Furlan A de O, Scaramele NF, Lopes FL, et al. Differential expression of miRNAs in canine peripheral blood mononuclear cells (PBMC) exposed to Leishmania infantum in vitro. Res Vet Sci. 2021;134: 58–63. doi:10.1016/j.rvsc.2020.11.021 130. Kumar V, Das S, Kumar A, Tiwari N, Kumar A, Abhishek K, et al. Leishmania donovani infection induce differential miRNA expression in CD4+ T cells. Sci Rep. 2020;10: 3523. doi:10.1038/s41598-020-60435-2 131. Pandey RK, Sundar S, Prajapati VK. Differential Expression of miRNA Regulates T Cell Differentiation and Plasticity During Visceral Leishmaniasis Infection. Front Microbiol. 2016;7: 1–9. doi:10.3389/fmicb.2016.00206 132. Varikuti S, Verma C, Natarajan G, Oghumu S, Satoskar AR. MicroRNA155 Plays a Critical Role in the Pathogenesis of Cutaneous Leishmania major Infection by Promoting a Th2 Response and Attenuating Dendritic Cell Activity. Am J Pathol. 2021;191: 809–816. doi:10.1016/j.ajpath.2021.01.012 133. Varikuti S, Verma C, Holcomb E, Jha BK, Viana A, Maryala R, et al. MicroRNA-21 Deficiency Promotes the Early Th1 Immune Response and Resistance toward Visceral Leishmaniasis. J Immunol. 2021;207: 1322– 1332. doi:10.4049/jimmunol.2001099 82 134. Melo LM, Bragato JP, Venturin GL, Rebech GT, Costa SF, Garcia LE, et al. Induction of miR 21 impairs the anti-Leishmania response through inhibition of IL-12 in canine splenic leukocytes. Satoskar AR, editor. PLoS One. 2019;14: e0226192. doi:10.1371/journal.pone.0226192 135. Muxel SM, Laranjeira-Silva MF, Zampieri RA, Floeter-Winter LM. Leishmania (Leishmania) amazonensis induces macrophage miR-294 and miR-721 expression and modulates infection by targeting NOS2 and L- arginine metabolism. Sci Rep. 2017;7: 44141. doi:10.1038/srep44141 136. Muxel SM, Acuña SM, Aoki JI, Zampieri RA, Floeter-Winter LM. Toll-Like Receptor and miRNA-let-7e Expression Alter the Inflammatory Response in Leishmania amazonensis-Infected Macrophages. Front Immunol. 2018;9: 2792. doi:10.3389/fimmu.2018.02792 137. Kumar V, Kumar A, Das S, Kumar A, Abhishek K, Verma S, et al. Leishmania donovani Activates Hypoxia Inducible Factor-1α and miR-210 for Survival in Macrophages by Downregulation of NF-κB Mediated Pro- inflammatory Immune Response. Front Microbiol. 2018;9: 385. doi:10.3389/fmicb.2018.00385 138. Nandan D, Rath CT, Reiner NE. Leishmania regulates host macrophage miRNAs expression by engaging transcription factor c-Myc. J Leukoc Biol. 2021;109: 999–1007. doi:10.1002/JLB.4RU0920-614R 139. Ghosh J, Bose M, Roy S, Bhattacharyya SN. Leishmania donovani Targets Dicer1 to Downregulate miR-122, Lower Serum Cholesterol, and Facilitate Murine Liver Infection. Cell Host Microbe. 2013;13: 277–288. doi:10.1016/j.chom.2013.02.005 140. Souza M de A, Ramos-Sanchez EM, Muxel SM, Lagos D, Reis LC, Pereira VRA, et al. miR-548d-3p Alters Parasite Growth and Inflammation in Leishmania (Viannia) braziliensis Infection. Front Cell Infect Microbiol. 2021;11: 1. doi:10.3389/fcimb.2021.687647 83 141. Ramos-Sanchez EM, Reis LC, Souza M de A, Muxel SM, Santos KR, Lagos D, et al. miR-548d-3p Is Up-Regulated in Human Visceral Leishmaniasis and Suppresses Parasite Growth in Macrophages. Front Cell Infect Microbiol. 2022;12: 110. doi:10.3389/fcimb.2022.826039 142. Di Loria A, Dattilo V, Santoro D, Guccione J, De Luca A, Ciaramella P, et al. Expression of Serum Exosomal miRNA 122 and Lipoprotein Levels in Dogs Naturally Infected by Leishmania infantum: A Preliminary Study. Animals. 2020;10: 100. doi:10.3390/ani10010100 143. Bragato JP, Melo LM, Venturin GL, Rebech GT, Garcia LE, Lopes FL, et al. Relationship of peripheral blood mononuclear cells miRNA expression and parasitic load in canine visceral Leishmaniasis. PLoS One. 2018;13: 1–16. doi:10.1371/journal.pone.0206876 144. Munshi SU, Panda H, Holla P, Rewari BB, Jameel S. MicroRNA-150 Is a Potential Biomarker of HIV/AIDS Disease Progression and Therapy. Sluis- Cremer N, editor. PLoS One. 2014;9: e95920. doi:10.1371/journal.pone.0095920 145. Shen J, Xing W, Gong F, Wang W, Yan Y, Zhang Y, et al. MiR-150-5p retards the progression of myocardial fibrosis by targeting EGR1. Cell Cycle. 2019;18: 1335–1348. doi:10.1080/15384101.2019.1617614 146. Akula SM, Bolin P, Cook PP. Cellular miR-150-5p may have a crucial role to play in the biology of SARS-CoV-2 infection by regulating nsp10 gene. RNA Biol. 2022;19: 1–11. doi:10.1080/15476286.2021.2010959 147. Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferon-γ: an overview of signals, mechanisms and functions. J Leukoc Biol. 2004;75: 163–189. doi:10.1189/jlb.0603252 148. Saadatian Z, Nariman-Saleh-Fam Z, Bastami M, Mansoori Y, Khaheshi I, Parsa SA, et al. Dysregulated expression of STAT1, miR-150, and miR- 84 223 in peripheral blood mononuclear cells of coronary artery disease patients with significant or insignificant stenosis. J Cell Biochem. 2019;120: 19810–19824. doi:10.1002/jcb.29286 149. Bian Y, Cai W, Lu H, Tang S, Yang K, Tan Y. miR-150-5p affects AS plaque with ASMC proliferation and migration by STAT1. Open Med. 2021;16: 1642–1652. doi:10.1515/med-2021-0357 150. Giunchetti RC, Silveira P, Resende LA, Leite JC, Melo-Júnior OA de O, Rodrigues- Alves ML, et al. Canine visceral leishmaniasis biomarkers and their employment in vaccines. Vet Parasitol. 2019;271: 87–97. doi:10.1016/j.vetpar.2019.05.006 151. Hu Z, Cui Y, Qiao X, He X, Li F, Luo C, et al. Silencing miR-150 Ameliorates Experimental Autoimmune Encephalomyelitis. Front Neurosci. 2018;12. doi:10.3389/fnins.2018.00465 152. Yao Y, Wang H, Xi X, Sun W, Ge J, Li P. miR-150 and SRPK1 regulate AKT3 expression to participate in LPS-induced inflammatory response. Innate Immun. 2021;27: 343–350. doi:10.1177/17534259211018800 153. Chakrabarti A, Oehme I, Witt O, Oliveira G, Sippl W, Romier C, et al. HDAC8: a multifaceted target for therapeutic interventions. Trends Pharmacol Sci. 2015;36: 481–492. doi:10.1016/j.tips.2015.04.013 154. Shakespear MR, Halili MA, Irvine KM, Fairlie DP, Sweet MJ. Histone deacetylases as regulators of inflammation and immunity. Trends Immunol. 2011;32: 335–343. doi:10.1016/j.it.2011.04.001 155. Haberland M, Montgomery RL, Olson EN. The many roles of histone deacetylases in development and physiology: implications for disease and therapy. Nat Rev Genet. 2009;10: 32–42. doi:10.1038/nrg2485 85 156. Goping IS, Barry M, Liston P, Sawchuk T, Constantinescu G, Michalak KM, et al. Granzyme B-Induced Apoptosis Requires Both Direct Caspase Activation and Relief of Caspase Inhibition. Immunity. 2003;18: 355–365. doi:10.1016/S1074-7613(03)00032-3 157. Smith NL, Wissink EM, Grimson A, Rudd BD. MiR-150 Regulates Differentiation and Cytolytic Effector Function in CD8+ T cells. Sci Rep. 2015. doi:10.1038/srep16399 102 ANEXO B - CERTIFICADO DO COMITÊ DE ÉTICA