Efeito das toxinas microbianas provenientes de biofilme simples ou misto de Staphylococcus aureus e Candida albicans sobre monoculturas ou culturas 3D de células da mucosa oral
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Data
2016-11-18
Autores
Dias, Kássia de Carvalho [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Esta presente tese foi dividida em quatro estudos que tiveram como objetivos. 1. Validar um protocolo e comparar o efeito dos tampões RPMI/MOPS e RPMI/HEPES no desenvolvimento de biofilmes e na viabilidade celular de queratinócitos (NOK-si e HaCat); 2. Comparar o dano celular e a resposta inflamatória induzidos pelos metabólitos de biofilmes simples e misto de Staphylococcus aureus e Candida albicans; 3. Avaliar o tipo de morte celular (apoptose vs. necrose) e a ativação de caspases relacionadas aos metabólitos desses biofilmes e 4. Caracterizar um tecido oral reconstituído e analisar o dano tecidual causado pelo sobrenadante e biofilme propriamente dito desses microrganismos. No estudo 1, a viabilidade celular foi avaliada pelo método colorimétrico do MTT e por imagens da cultura após 12 horas em contato com os meios de cultura. Ambos os tampões permitiram similar crescimento do biofilme. Efeito citotóxico do MOPS foi verificado após 6 horas de crescimento de NOK-si e HaCat. Houve preservação da viabilidade e morfologia quando as células foram expostas a RPMI/HEPES. Conclui-se que RPMI/HEPES pode ser utilizado como um meio tamponamente viável para estudos que avaliam o efeito do biofilme em cultura de queratinócitos ao longo do tempo. No estudo 2, o sobrenadante dos biofilmes de 36 h de C. albicans e S. aureus, isolados ou em associação, foi colocado em contato com NOK-si, HaCat e macrófagos (J774A.1). O dano celular foi avaliado por meio de ensaios de viabilidade celular (MTT) e liberação da enzima LDH. A produção de citocinas foi analisada pelo método de ELISA e a avaliação do tipo de morte celular pela marcação das células apoptóticas com anexina V e necróticas com iodeto de propídio. Biofilme misto e de C. albicans foram mais citotóxicos e o biofilme misto causou maior dano celular pela liberação da enzima LDH. Metabólitos do biofilme de S. aureus estimularam maior produção de NO, IL-6 e TNF-α. Maior quantidade de células marcadas com anexina V (apoptose) foram detectadas após exposição ao sobrenadante de C. albicans, e maior marcação para o iodeto de propídio (necrose) para as células expostas ao sobrenadante do biofilme misto. Os metabólitos de biofilmes mistos foram capazes de induzir maior dano celular e os metabólitos de S. aureus, maior resposta inflamatória. No estudo 3, a indução de morte celular em cultura de NOK-si foi avaliada após o contato com sobrenadante de biofilmes simples ou mistos, com diferentes tempos de formação (08, 16 e 24 horas). As células microbianas foram quantificadas em UFC/mL e os danos aos queratinócitos foram avaliados pelos ensaios de MTT e de liberação da enzima LDH. Houve marcação de apoptose e necrose como no estudo anterior e ensaio colorimétrico para detecção dos marcadores de apoptose celular (caspases). A morfologia celular dos queratinócitos foi avaliada por meio de microscopia eletrônica de varredura (MEV). Houve um aumento progressivo nos valores de UFC/mL obtidos dos biofilmes de 08 horas para 16 horas, tanto para os biofilmes simples, quanto para os mistos. Houve uma redução progressiva na viabilidade celular após exposição ao biofilme de 16 horas quando comparado ao biofilme de 08 e 24 horas. As imagens de MEV mostraram uma diminuição das microvilosidades e a formação de vesículas na membrana das células expostas ao sobrenadante do biofilme de 16 horas. Houve também maior marcação de anexina V quando as células foram expostas ao sobrenadante de biofilmes simples de C. albicans e S. aureus; e com iodeto de propídio para o biofilme misto. A apoptose induzida pelos sobrenadantes do biofilme de S. aureus e misto foi associada à ativação das caspases -2, -3, -6 e -8; e o aumento da atividade da caspase -3 para sobrenadante de C. albicans. Assim, maior dano celular pode ser observado quando queratinócitos foram expostos a metabólitos dos microrganismos em sinergismo no biofilme. No estudo 4, tecido oral reconstituído foi obtido pela formação in vitro de um equivalente de tecido conjuntivo (fibroblastos e colágeno) e uma camada epitelial (queratinócitos). A caracterização histológica foi obtida pelas colorações de Hematoxilina-Eosina, Ácido Periódico de Schiff - P.A.S. e Picrosirius e a caracterização imunohistoquímica pela evidenciação de citoqueratina 13 e 14, Ki-67 e colágeno IV. A análise do dano causado ao tecido pelo sobrenadante e biofilmes, com 08 e 16 horas, foi observada por meio da liberação da enzima LDH e avaliação histológica (Hematoxilina-Eosina). O biofilme misto foi responsável pela maior destruição tecidual. O tecido epitelial reconstituído apresentou características histológicas e imunohistoquímicas semelhantes ao epitélio oral e pode ser utilizado para investigar padrões de infecção causada por biofilmes em culturas simples e mista de C. albicans e S. aureus.
The present thesis was divided into four studies with the following objectives. 1. Validate a protocol and compare the effect of RPMI/MOPS and RPMI/HEPES buffers on the development of biofilms and keratinocyte cell viability (NOK-si and HaCat); 2. Compare the cellular damage and the inflammatory response induced by the metabolites of simple and mixed biofilms of Staphylococcus aureus and Candida albicans; 3. Evaluate the type of cell death (apoptosis vs. necrosis) and the activation of caspases related to the metabolites of these biofilms and 4. Characterize the reconstituted oral tissue and analyze the tissue damage caused by the supernatant and biofilm of these microorganisms. In study 1, cell viability was evaluated by the MTT colorimetric method and by culture images after 12 hours in contact with the culture media. Both buffers permitted similar biofilm growth. The cytotoxic effect of MOPS was observed after six hours of NOK-si and HaCat growth. There was preservation of viability and morphology when cells were exposed to RPMI/HEPES. It was concluded that RPMI/HEPES can be used as a buffering medium for studies evaluating the effect of biofilm on keratinocyte culture over time. In study 2, the supernatant of the 36-hour biofilms of C. albicans and S. aureus, isolated or in combination, was placed in contact with NOK-si, HaCat and macrophages (J774A.1). Cell damage was assessed by cell viability assays (MTT) and LDH enzyme release. Cytokine production was analyzed by the ELISA method and evaluation of the type of cell death by the staining of the apoptotic cells with annexin V and the necrotic cells with propidium iodide. The mixed biofilm and biofilm of C. albicans were more cytotoxic, and the mixed biofilm caused greater cellular damage through the release of the LDH enzyme. S. aureus biofilm metabolites stimulated greater production of NO, IL-6 and TNF-α. Greater numbers of cells labeled with annexin V (apoptosis) were detected after exposure to the supernatant of C. albicans, and there was greater propidium iodide staining (necrosis) for cells exposed to the supernatant of the mixed biofilm. The mixed biofilm metabolites were able to induce greater cellular damage and the metabolites of S. aureus produced a greater inflammatory response. In study 3, cell death induction in NOK-si culture was evaluated after contact with the supernatant of single or mixed biofilms, with different formation times (08, 16 and 24 hours). Microbial cells were quantified in terms of CFU/mL and damage to keratinocytes was assessed by MTT assay and LDH enzyme release. Apoptosis and necrosis staining was evaluated as in the previous study and colorimetric assay was performed for the detection of cellular apoptosis markers (caspases). The cellular morphology of the keratinocytes was evaluated by scanning electron microscopy (SEM). There was a progressive increase in the CFU/mL values obtained from biofilms from eight to 16 hours for both single and mixed biofilms. There was a progressive reduction in cell viability after exposure to the 16-hour biofilm in comparison with the 08 and 24-hour biofilm. SEM images exhibited a decrease in microvilli and the formation of vesicles on the membrane of the cells exposed to the supernatant of the 16-hour biofilm. There was also greater annexin V staining when the cells were exposed to the supernatant of the single biofilms of C. albicans and S. aureus; and greater propidium iodide staining with the mixed biofilm. Apoptosis induced by supernatants of S. aureus biofilm and mixed biofilm was associated with the activation of caspases -2, -3, -6 and -8, while increased caspase -3 activity was associated with the C. albicans supernatant. Thus, greater cellular damage can be observed when keratinocytes were exposed to the metabolites of the microorganisms in synergism in biofilm. In study 4, reconstituted oral tissue was obtained by the in vitro formation of an equivalent of connective tissue (fibroblasts and collagen) and an epithelial layer (keratinocytes). Histological characterization was performed by Hematoxylin-Eosin, Periodic Acid Schiff - P.A.S. and Picrosirius staining, and immunohistochemical characterization by cytokeratin 13 and 14, Ki-67 and collagen IV. The analysis of the tissue damage caused by the supernatant and biofilms at eight and 16 hours was observed through the release of the LDH enzyme and histological evaluation (Hematoxylin-Eosin). The mixed biofilm was responsible for the greatest tissue destruction. The reconstituted epithelial tissue presented histological and immunohistochemical characteristics similar to the oral epithelium and can be used to investigate infection patterns caused by biofilms of C. albicans and S. aureus in single and mixed cultures.
The present thesis was divided into four studies with the following objectives. 1. Validate a protocol and compare the effect of RPMI/MOPS and RPMI/HEPES buffers on the development of biofilms and keratinocyte cell viability (NOK-si and HaCat); 2. Compare the cellular damage and the inflammatory response induced by the metabolites of simple and mixed biofilms of Staphylococcus aureus and Candida albicans; 3. Evaluate the type of cell death (apoptosis vs. necrosis) and the activation of caspases related to the metabolites of these biofilms and 4. Characterize the reconstituted oral tissue and analyze the tissue damage caused by the supernatant and biofilm of these microorganisms. In study 1, cell viability was evaluated by the MTT colorimetric method and by culture images after 12 hours in contact with the culture media. Both buffers permitted similar biofilm growth. The cytotoxic effect of MOPS was observed after six hours of NOK-si and HaCat growth. There was preservation of viability and morphology when cells were exposed to RPMI/HEPES. It was concluded that RPMI/HEPES can be used as a buffering medium for studies evaluating the effect of biofilm on keratinocyte culture over time. In study 2, the supernatant of the 36-hour biofilms of C. albicans and S. aureus, isolated or in combination, was placed in contact with NOK-si, HaCat and macrophages (J774A.1). Cell damage was assessed by cell viability assays (MTT) and LDH enzyme release. Cytokine production was analyzed by the ELISA method and evaluation of the type of cell death by the staining of the apoptotic cells with annexin V and the necrotic cells with propidium iodide. The mixed biofilm and biofilm of C. albicans were more cytotoxic, and the mixed biofilm caused greater cellular damage through the release of the LDH enzyme. S. aureus biofilm metabolites stimulated greater production of NO, IL-6 and TNF-α. Greater numbers of cells labeled with annexin V (apoptosis) were detected after exposure to the supernatant of C. albicans, and there was greater propidium iodide staining (necrosis) for cells exposed to the supernatant of the mixed biofilm. The mixed biofilm metabolites were able to induce greater cellular damage and the metabolites of S. aureus produced a greater inflammatory response. In study 3, cell death induction in NOK-si culture was evaluated after contact with the supernatant of single or mixed biofilms, with different formation times (08, 16 and 24 hours). Microbial cells were quantified in terms of CFU/mL and damage to keratinocytes was assessed by MTT assay and LDH enzyme release. Apoptosis and necrosis staining was evaluated as in the previous study and colorimetric assay was performed for the detection of cellular apoptosis markers (caspases). The cellular morphology of the keratinocytes was evaluated by scanning electron microscopy (SEM). There was a progressive increase in the CFU/mL values obtained from biofilms from eight to 16 hours for both single and mixed biofilms. There was a progressive reduction in cell viability after exposure to the 16-hour biofilm in comparison with the 08 and 24-hour biofilm. SEM images exhibited a decrease in microvilli and the formation of vesicles on the membrane of the cells exposed to the supernatant of the 16-hour biofilm. There was also greater annexin V staining when the cells were exposed to the supernatant of the single biofilms of C. albicans and S. aureus; and greater propidium iodide staining with the mixed biofilm. Apoptosis induced by supernatants of S. aureus biofilm and mixed biofilm was associated with the activation of caspases -2, -3, -6 and -8, while increased caspase -3 activity was associated with the C. albicans supernatant. Thus, greater cellular damage can be observed when keratinocytes were exposed to the metabolites of the microorganisms in synergism in biofilm. In study 4, reconstituted oral tissue was obtained by the in vitro formation of an equivalent of connective tissue (fibroblasts and collagen) and an epithelial layer (keratinocytes). Histological characterization was performed by Hematoxylin-Eosin, Periodic Acid Schiff - P.A.S. and Picrosirius staining, and immunohistochemical characterization by cytokeratin 13 and 14, Ki-67 and collagen IV. The analysis of the tissue damage caused by the supernatant and biofilms at eight and 16 hours was observed through the release of the LDH enzyme and histological evaluation (Hematoxylin-Eosin). The mixed biofilm was responsible for the greatest tissue destruction. The reconstituted epithelial tissue presented histological and immunohistochemical characteristics similar to the oral epithelium and can be used to investigate infection patterns caused by biofilms of C. albicans and S. aureus in single and mixed cultures.
Descrição
Palavras-chave
Biofilmes, Queratinócitos, Candida albicans, Staphylococcus aureus, Necrose, Apoptose, Biofilms, Keratinocytes, Necrosis, Apoptosis