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Doctoral Thesis
DOI
10.11606/T.5.2016.tde-06062016-115609
Document
Author
Full name
Rosana Domingues Parron
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2016
Supervisor
Committee
Sato, Maria Notomi (President)
Aoki, Valeria
Oliveira, Ana Paula Ligeiro de
Sanches Junior, Jose Antonio
Tomimori, Jane
Title in Portuguese
Análise fenotípica de células dendríticas e de linfócitos T efetores, polifuncionais e reguladores no líquen plano
Keywords in Portuguese
Células dendríticas
Citocinas
Linfócitos T
Linfócitos T reguladores
Líquen plano/imunologia
Receptores Toll-like
Abstract in Portuguese
INTRODUÇÃO: Líquen plano (LP) é uma doença mucocutânea de natureza inflamatória crônica de etiologia ainda desconhecida. A estimulação da imunidade inata via os receptores Toll-like (TLRs) podem influenciar as células dendríticas e direcionar a resposta de células T CD4+ e CD8+ efetoras, assim como também favorecer o estado inflamatório do LP. OBJETIVOS: Avaliar o perfil fenotípico de células dendríticas mielóides (mDCs) e plasmocitóides (pDCs) e de linfócitos T CD4+ e CD8+ após estímulo com agonistas de TLRs no sangue periférico de pacientes com LP. Além disto, avaliar a frequência, perfil de maturação e os subtipos de células T CD4+ e TCD8+ reguladores. MÉTODOS: Foram selecionados 18 pacientes com LP (15 mulheres, 3 homens), com 41,57 ± 4,73 anos de idade e um grupo controle com 22 indivíduos sadios (18 mulheres, 4 homens), com 43,92 ± 7,83 anos de idade. As células mononucleares (CMNs) de sangue periférico foram avaliadas por citometria de fluxo quanto à: 1) Produção de TNF-? em mDCs e de IFN-? em pDCs em CMNs ativadas por agonistas de TLR 4, 7, 7/8 e 9; 2) Análise de células T CD4+ e CD8+ monofuncionais e polifuncionais após estímulo com agonistas de TLR 4, 7/8, 9 e enterotoxina B de Staphylococcus aureus (SEB); 3) Avaliação de células Th17 e Th22/Tc22 em CMNs após estímulo com SEB; 4) Frequência, perfil de maturação e subtipos de células T CD4+ e CD8+ reguladoras. RESULTADOS: 1) Nos pacientes com LP foi demonstrado um aumento na frequência de mDCs TNF-alfa+ após estímulo com agonistas de TLR4/LPS e TLR7-8/CL097, mas com imiquimode/TLR7 houve diminuição da expressão de CD83. Já nas pDCs do grupo LP, o imiquimode foi capaz de diminuir a expressão de CD80 e o CpG/TLR9 diminuiu a expressão de CD83 no LP. 2) As células T CD4+ secretoras de IL-10 mostraram aumento da frequência nos níveis basais, que diminuiu após estímulo com LPS e SEB. Em contraste, a produção de IFN-y aumentou em resposta ao LPS enquanto diminuiu para CpG. As células T CD4+ polifuncionais, secretoras de 5 citocinas simultâneas (CD4+IL-17+IL-22+TNF+IL-10+IFN-y+) diminuíram no LP após estímulo com CL097 e CpG. Entretanto, na ausência de IL-10, houve aumento da frequência de células CD4+IL-17+IL-22+TNF+IFN-y+ em resposta ao LPS. Um aumento na polifuncionalidade foi observado em células TCD4+ que expressam CD38, marcador de ativação crônica e na ausência de IL-10. Similarmente, às TCD4+, uma diminuição de células T CD8+ IFN-y+ e TNF+ foram detectadas após estímulo com CpG. 3) As células Th22/Tc22 nos níveis basais e após estímulo com SEB se mostraram aumentadas. As células Th17 não mostraram diferenças entre os grupos. 4) A frequência das células T CD4+ e CD8+ reg totais (CD25+Foxp3+CD127low/-) está elevada no LP. Quanto aos perfis de maturação, há aumento na frequência de células TCD4+ de memória efetora enquanto que para as células T CD8+ há predomínio das células de memória central. Quanto aos subtipos, há aumento nas células T CD4+ regs periféricas (pT reg). CONCLUSÕES: O estado de ativação das mDCs após ativação das vias de TLRs 4 e 7/8 pode influenciar na geração de resposta T efetoras no LP. O perfil de resposta monofuncional e polifuncional aos estímulos TLRs reflete a ativação destas células no sangue periférico. Além disso, o aumento de Th22/Tc22 e das células T regs indicam uma relação entre regulação e células efetoras no sangue periférico evidenciando que existem alterações extracutâneas no LP
Title in English
Phenotypic analysis of dendritic cells and effector, polyfunctional and regulators lymphocytes on lichen planus
Keywords in English
Cytokines
Dendritic cells, T-lymphocytes
Lichen planus/immunology
T-lymphocytes regulatory
Toll-like receptors
Abstract in English
BACKGROUND: Lichen planus (LP) is a mucocutaneous disease of chronic inflammatory course of unknown etiology. Stimulation of the innate immune system via Toll-like receptors (TLRs) may influence the dendritic cells and targeting the CD4+ and effector CD8+ T cell responses, as well as promoting inflammatory status of the LP. OBJECTIVES: To evaluate the phenotypic profile of myeloid dendritic cells (mDCs), plasmacytoid (pDCs) and CD4+ and CD8+ T lymphocytes after stimulation with TLR agonists in peripheral blood of patients with LP. Moreover, to evaluate the frequency, maturation profile and subtypes of CD4+ and CD8+ T regulators cells. METHODS: We selected 18 patients with LP (15 women, 3 men) with 41.57 ± 4.73 years old and a control group of 22 healthy subjects (18 women, 4 men), with 43.92 ± 7, 83 years old. Mononuclear cells from peripheral blood (PBMCs) were assessed by flow cytometry for: 1) mDC TNF-alfa production and pDCs IFN-alfa production in PBMCs activated by agonists of TLR 4, 7, 7/8 and 9; 2) Analysis of monofunctional and polyfunctional CD4+ and CD8+ T cells after stimulation with TLR 4 agonists, 7/8, 9 and Staphylococcus aureus enterotoxin B (SEB); 3) Evaluation of Th17 and Th22/ Tc22 cells in PBMCs after stimulation with SEB; 4) Frequency, maturation profile and subtypes of regulatory CD4+ and CD8+ T cells. RESULTS: 1) Patients with LP showed an increased frequency of TNF-alfa+ mDCs after stimulation with agonists of TLR4/LPS and TLR7-8 /CL097, whereas with imiquimod /TLR7 induced a decreased CD83 expression. Already in the pDCs of LP group the imiquimod was able to decrease the CD80 expression and CpG/TLR9 decreased CD83 expression. 2) CD4+ T cells secreting IL-10 demonstrated an increased frequency at the baseline levels, which decreased after stimulation with LPS and SEB. In contrast, the production of IFN-y increased in response to LPS while decreased to CpG. Polyfunctional CD4+ T cells secreting simultaneously 5 cytokines (CD4+IL-17+IL-22+TNF+IL-10+IFN-y+) decreased in the LP after stimulation with CpG and CL097. However, in the absence of IL-10, occurred an increased frequency of CD4+IL-17+IL-22+IFN-y+TNF+ in response to LPS. An increase in the polyfunctional response was seems in CD4+ T cells expressing CD38, a chronic activation marker, in the absence of IL-10. Similarly to the CD4+ T cells, a decreased CD8+ T cells secreting IFN-? and TNF was observed in LP, after stimulation with CpG. Polyfunctional CD8+ T cells from LP group showed decreased response with 3 and 4 cytokines at baseline condition, and upon SEB and CL097 stimulations, occurred an increased frequency of these cells. In LP group, T cells CD8+CD38+ polyfunctional showed low capacity, such as CD4+CD38+ cells. 3) The Th22/Tc22 cells already at baseline and after stimulation with SEB showed increased frequency. The Th17 cells showed no differences between groups. 4) The frequency of CD4+ and CD8+ total reg (CD25+Foxp3+CD127low/-) is increased in LP. The profiles of maturity, an increase in the frequency of CD4+ effector cells whereas for memory CD8+ T cells there is a predominance of central memory cells. As for the subtypes, there is an increase in CD4+ peripheral T regs cells (pT reg). CONCLUSIONS: The activation state of mDCs after activation of the pathways of TLRs 4 and 7/8 can influence the response and generation of effector T cells in the LP. The monofunctional and polyfunctional response profile for TLRs stimuli reflects the activation of these cells in peripheral blood. Furthermore, the increase of Th22/Tc22 and T regs cells indicate a relationship between regulatory and effector cells in peripheral blood showing that there are extra-cutaneous alterations in the LP
 
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Publishing Date
2016-06-06
 
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