PD-L1 protein inhibition in a murine model of immediate hypersensitivity

INTRODUÇÃO: Os mastócitos atuam no sistema de defesa contra patógenos e no desenvolvimento de distúrbios alérgicos. Embora as respostas dependentes de IgE via Fc-Épsilon-RI nessas células tenham sido amplamente estudadas, pouco se sabe sobre como as moléculas de superfície celular, como o PD-1 e seus ligantes, que são expressas por mastócitos, células dendríticas e linfócitos T podem modular a reação alérgica. O presente estudo analisou os efeitos do bloqueio da molécula PD-L1 em um modelo murino de anafilaxia cutânea. MÉTODOS: Camundongos C57BL/6 foram sensibilizados e desafiados com ovalbumina segundo protocolo que teve duração de 28 dias. Amostras de sangue foram coletadas nos dias 0, 14 e 28 para dosagem de IgE, IgG1 e IgG2a específicos. Os camundongos foram divididos em 9 grupos. Desses grupos, 6 foram desafiados com a técnica de anafilaxia cutânea ativa. Dos seis grupos, um grupo recebeu por via subcutânea 50 Micro l de tampão fosfato-salino e os outros 5 grupos foram sensibilizados por via subcutânea com 50 Micro g de ovalbumina, 1 vez por semana, durante quatro semanas. Desses 5 grupos sensibilizados à ovalbumina, o primeiro grupo recebeu anticorpo anti-PD-L1 e o segundo recebeu o isotipo do anticorpo anti-PD-L1 durante a sensibilização, o terceiro recebeu anticorpo anti-PD-L1 e o quarto recebeu o isotipo do anticorpo anti-PD-L1 durante o desafio e o quinto grupo foi o grupo controle. Esses 6 grupos desafiados com a técnica de anafilaxia cutânea ativa foram provocados na orelha com injeção subcutânea de 10 Micro l de ovalbumina a 5 Micro g/Micro l, além de receberem 200 Micro l de azul de Evans a 0,025% por via intravenosa. Após 20 minutos, os animais foram sacrificados e a reação foi avaliada pelo extravasamento de azul de Evans (mensurado por espectrofotometria) e análise histológica dos fragmentos coletados. Os 3 grupos de camundongos restantes foram destinados para o desafio com a técnica de anafilaxia cutânea passiva, sendo sensibilizados com soro de camundongos previamente sensibilizados à ovalbumina em diferentes diluições (1/40, 1/80, 1/160 e 1/320). Desses 3 grupos, o primeiro grupo recebeu anticorpo anti-PD-L1, o segundo recebeu o isotipo do anticorpo anti-PD-L1 e o terceiro foi o grupo controle. Azul de Evans 200 Micro l 0,25% e 10 Micro l do alérgeno ovalbumina a 5 Micro g/Micro l foram injetados por via intravenosa. Após 20 minutos, os animais foram sacrificados e a reação foi avaliada pelo extravasamento de azul de Evans e análise histológica de fragmentos coletados. RESULTADOS: Os níveis séricos de IgE e IgG1 específicas foram significativamente maiores, nos dias 14 e 28 no grupo controle positivo, não tratado com anticorpos, e isotipo anti-PD-L1 do que no grupo que recebeu anti-PD-L1 durante a sensibilização. Houve redução significativa no extravasamento de azul Evans no grupo que recebeu anti-PD-L1 durante a sensibilização quando comparado ao controle positivo e ao grupo isotipo anti-PD-L1. Não houve variação significativa entre os grupos nos experimentos de anafilaxia cutânea passiva. CONCLUSÃO: O bloqueio da molécula PD-L1 durante a sensibilização com alérgeno inibe a síntese de IgE e a IgG1 específicas e diminui a resposta alérgicaINTRODUCTION: Mast cells are an important part of the innate immunity system and participate in the defense against pathogens, but are also involved in the development of allergic disorders. Although IgE-dependent immune responses via Fc Epsilon RI have been extensively studied, it is not know if immediate hypersensitivity can be modulated by surface molecules such as PD-1 and its ligand PD-L1, which are expressed on mast cells, dendritic cells and T lymphocytes and have been shown to have an inhibitory role in cancer. The present study assessed the effects of PD-L1 blockage in a murine model of cutaneous anaphylaxis. METHODS: C57BL/6 mice were sensitized and challenged with ovalbumin according to a protocol that lasted 28 days. Blood samples were collected on days 0, 14 and 28 for IgE, IgG1 and IgG2a measurements. Mice were divided into 9 groups, with 5 mice per group. Of these groups, 6 were challenged through an active cutaneous anaphylaxis protocol. Of the six groups, one group received 50 Micro l phosphate-saline buffer subcutaneously and the other 5 groups were sensitized subcutaneously with 50 Micro g ovalbumin once weekly for four weeks. Of these 5 groups sensitized to ovalbumin, the first and second groups received anti-PD-L1 antibody or isotype control during sensitization, the third and fourth received anti-PD-L1 antibody or isotope control during challenge, the fifth group was the positive control group sensitized and challenge to ovalbumin and the sixth group was the negative control group that was not sensitized to ovalbumin but it was challenged to ovalbumin. These 6 groups underwent active cutaneous anaphylaxis protocols and were challenged in the ear with subcutaneous injections of 10 Micro l of 5 Micro g/Micro l Ovalbumin and Evans blue, 200 Micro l, 0.025%, was administered intravenously. After 20 minutes, the animals were sacrificed and the allergic reaction was evaluated by Evans blue extravasation (measured by spectrophotometry) and histological analysis of the fragments collected. The remaining 3 groups were assigned to the passive cutaneous anaphylaxis protocol, where mice were sensitized with serum from previously sensitized animals to ovalbumin at different dilutions (1/40, 1/80, 1/160 and 1/320). Of these 3 groups, the first and second group received anti-PD-L1 antibody and anti-PD-L1 antibody isotype, respectively, during challenge and the third was the positive control group (please explain the positive control). Evans blue 200 Micro l 0.25% and 10 Micro l of ovalbumin allergen at 5 Micro g/Micro l were injected intravenously. After 20 minutes, the animals were sacrificed and the reaction was evaluated by Evans blue extravasation and histological analysis of collected fragments. RESULTS: Specific serum IgE and IgG1 levels were significantly higher on days 14 and 28 in the positive control and anti-PD-L1 isotype groups than in the group that received anti-PD-L1 during sensitization. There was a significant reduction in the extravasation of Evans blue in the group that received anti-PD-L1 during sensitization when compared to both the positive control and anti-PD-L1 isotype groups. In contrast subcutaneous challenge with allergen on the animals back in the passive anaphylaxis protocol did not show statistically significant differences between the groups indicating that PD-L1 modulation of the sensitization phase could account for the differences seen during active anaphylaxis. CONCLUSION: Blocking of the PD-L1 molecule during allergen sensitization inhibits specific IgE and IgG1 synthesis and decreases the allergic respons