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Authors
Abstract(s)
Os recetores do tipo Toll (TLR) são componentes do sistema imunitário inato, responsáveis
pelo reconhecimento de padrões moleculares associados a agentes patogénicos (PAMPs) e
pelo início de respostas imunitárias. Este estudo tem como objetivo estudar a ativação
diferencial dos heterodímeros TLR2 por ligandos específicos e a ativação de vias de
sinalização a jusante em diferentes linhas celulares.
Utilizámos células repórter HEK-Blue concebidas para expressar especificamente TLR2/1,
TLR2/6 ou TLR4, de modo a avaliar as suas respostas funcionais a extratos de Escherichia
coli (HKEB), e de Staphylococcus aureus (HKSA) preparados a partir de bactérias mortas pelo
calor. A análise de Western blot demonstrou padrões de expressão distintos das proteínas
adaptadoras TRIF e My-D88, o que mostra a adequada utilização das linhas celulares
selecionadas para estudar a sinalização dos recetores do tipo Toll. Posteriormente, os nossos
resultados indicaram que os heterodímeros TLR2/1 apresentaram maior atividade em
resposta a HKEB, enquanto os heterodimeros TLR2/6 foram preferencialmente ativados por
HKSA. Adicionalmente, o TLR4 foi exclusivamente responsivo à HKEB, confirmando o seu
papel como recetor primário a componentes bacterianos Gram-negativos. Nos queratinócitos
HaCaT, observámos um aumento significativo na secreção de IL-8 após estimulação com
HKSA, particularmente em concentrações mais elevadas, sugerindo que estas células são
mais sensíveis a componentes bacterianos Gram-positivos.
Os nossos resultados mostram que os modelos in vitro escolhidos podem ser utilizados para
o rastreio de ligandos específicos para os diferentes TLR com o objetivo de ajudar a melhorar
a nossa compreensão acerca da especificidade da sinalização TLR e das funções únicas de
TRIF e MYD88 em diferentes contextos celulares. Estes resultados têm implicações
significativas para o desenvolvimento de imunoterapias e vacinas direcionadas para agentes
patogénicos específicos. Embora o nosso estudo ofereça um contributo para este domínio,
são essenciais estudos in vivo para validar estes mecanismos.
Em resumo, esta investigação elucida a complexa interação entre os TLR e as suas proteínas
adaptadoras na formação de respostas imunitárias a agentes patogénicos bacterianos,
lançando as bases para futuros estudos destinados a melhorar as estratégias terapêuticas em
doenças infeciosas.
Toll-like receptors are integral components of the innate immune system, responsible for recognizing pathogen-associated molecular patterns (PAMPs) and initiating immune responses. This study aims to investigate the differential activation of TLR2 heterodimers by specific ligands, and the activation of downstream signaling pathways across various cell types. We utilized HEK-Blue reporter cells engineered to specifically express TLR2/1, TLR2/6 or TLR4, to assess their functional responses to heat-killed Escherichia coli (HKEB) and Staphylococcus aureus (HKSA). Western blot analysis demonstrated distinct expression patterns of TRIF and My-D88 adaptor proteins, which show the adequacy of the chosen cell lines, to study TLR signaling. Afterwards, our results indicated that TLR2/1 heterodimers showed higher activity in response to HKEB, while TLR2/6 receptors were preferentially activated by HKSA. Notably, TLR4 was exclusively responsive to HKEB, confirming its role as a primary receptor for Gram-negative bacterial components. In HaCaT keratinocytes, we observed a significant increase in IL-8 secretion upon stimulation with HKSA, particularly at higher concentrations, suggesting that these cells are more responsive to Gram-positive bacterial components. This IL-8 response is indicative of TLR2-mediated signaling, emphasizing the skin's innate immune capacity to respond to pathogens commonly associated with skin infections. Our results show that the in vitro models chosen, can be used for high-throughput screening of specific TLR ligands, and will help to enhance our understanding of TLR signaling specificity and the unique functions of TRIF and MYD88 in different cellular contexts. Moreover, these findings have significant implications for the development of targeted immunotherapies and vaccines to specific pathogens. While the study offers valuable contributions to the field, further in vivo investigations are essential to validate these mechanisms and refine our understanding of TLR signaling dynamics across diverse tissue types and environments. In summary, this research elucidates the complex interplay between TLRs and their adaptor proteins in shaping immune responses to bacterial pathogens, laying the groundwork for future studies aimed at improving therapeutic strategies in infectious diseases.
Toll-like receptors are integral components of the innate immune system, responsible for recognizing pathogen-associated molecular patterns (PAMPs) and initiating immune responses. This study aims to investigate the differential activation of TLR2 heterodimers by specific ligands, and the activation of downstream signaling pathways across various cell types. We utilized HEK-Blue reporter cells engineered to specifically express TLR2/1, TLR2/6 or TLR4, to assess their functional responses to heat-killed Escherichia coli (HKEB) and Staphylococcus aureus (HKSA). Western blot analysis demonstrated distinct expression patterns of TRIF and My-D88 adaptor proteins, which show the adequacy of the chosen cell lines, to study TLR signaling. Afterwards, our results indicated that TLR2/1 heterodimers showed higher activity in response to HKEB, while TLR2/6 receptors were preferentially activated by HKSA. Notably, TLR4 was exclusively responsive to HKEB, confirming its role as a primary receptor for Gram-negative bacterial components. In HaCaT keratinocytes, we observed a significant increase in IL-8 secretion upon stimulation with HKSA, particularly at higher concentrations, suggesting that these cells are more responsive to Gram-positive bacterial components. This IL-8 response is indicative of TLR2-mediated signaling, emphasizing the skin's innate immune capacity to respond to pathogens commonly associated with skin infections. Our results show that the in vitro models chosen, can be used for high-throughput screening of specific TLR ligands, and will help to enhance our understanding of TLR signaling specificity and the unique functions of TRIF and MYD88 in different cellular contexts. Moreover, these findings have significant implications for the development of targeted immunotherapies and vaccines to specific pathogens. While the study offers valuable contributions to the field, further in vivo investigations are essential to validate these mechanisms and refine our understanding of TLR signaling dynamics across diverse tissue types and environments. In summary, this research elucidates the complex interplay between TLRs and their adaptor proteins in shaping immune responses to bacterial pathogens, laying the groundwork for future studies aimed at improving therapeutic strategies in infectious diseases.
Description
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2024, Universidade de Lisboa, Faculdade de Farmácia.
Keywords
Innate immunity Toll-like receptors Immune signaling pathways HEK-Blue reporter cells Mestrado Integrado - 2024