携带编码IL-4的pValac真核表达载体的乳酸乳球菌，通过增加产生IL-10的调节性细胞水平来减轻化学诱导的肠道炎症。

Lactococcus lactis carrying the pValac eukaryotic expression vector coding for IL-4 reduces chemically-induced intestinal inflammation by increasing the levels of IL-10-producing regulatory cells.

作者信息

Souza Bianca Mendes, Preisser Tatiane Melo, Pereira Vanessa Bastos, Zurita-Turk Meritxell, de Castro Camila Prósperi, da Cunha Vanessa Pecini, de Oliveira Rafael Pires, Gomes-Santos Ana Cristina, de Faria Ana Maria Caetano, Machado Denise Carmona Cara, Chatel Jean-Marc, Azevedo Vasco Ariston de Carvalho, Langella Philippe, Miyoshi Anderson

机构信息

Laboratório de Tecnologia Genética, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

Departamento de Ciências Biológicas, Instituto Federal do Paraná, Foz do Iguaçu, Paraná, Brazil.

出版信息

Microb Cell Fact. 2016 Aug 30;15(1):150. doi: 10.1186/s12934-016-0548-x.

DOI:10.1186/s12934-016-0548-x

PMID:27576902

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5006358/

Abstract

BACKGROUND

Inflammatory bowel diseases are characterized by chronic intestinal inflammation that leads to severe destruction of the intestinal mucosa. Therefore, the understanding of their aetiology as well as the development of new medicines is an important step for the treatment of such diseases. Consequently, the development of Lactococcus lactis strains capable of delivering a eukaryotic expression vector encoding the interleukin 4 (IL-4) of Mus musculus would represent a new strategy for the elaboration of a more effective alternative therapy against Crohn's disease.

RESULTS

The murine IL-4 ORF was cloned into the eukaryotic expression vector pValac::dts. The resulting plasmid-pValac::dts::IL-4-was transfected into CHO cells so that its functionality could be evaluated in vitro. With fluorescent confocal microscopy, flow cytometry and ELISA, it was observed that pValac::dts::IL-4-transfected cells produced IL-4, while non-transfected cells and cells transfected with the empty vector did not. Then, pValac::dts::IL-4 was inserted into L. lactis MG1363 FnBPA(+) in order to evaluate the therapeutic potential of the recombinant strain against TNBS-induced colitis. Intragastric administration of L. lactis MG1363 FnBPA(+) (pValac::dts::IL-4) was able to decrease the severity of colitis, with animals showing decreased levels of IL-12, IL-6 and MPO activity; and increased levels of IL-4 and IL-10. Finally, LP-isolated cells from mice administered TNBS were immunophenotyped so that the main IL-4 and IL-10 producers were identified. Mice administered the recombinant strain presented significantly higher percentages of F4/80(+)MHCII(+)Ly6C(-)IL-4(+), F4/80(+)MHCII(+)Ly6C(-)IL-10(+), F4/80(+)MHCII(+)Ly6C(-)CD206(+)CD124(+)IL-10(+) and CD4(+)Foxp3(+)IL10(+) cells compared to the other groups.

CONCLUSIONS

This study shows that L. lactis MG1363 FnBPA(+) (pValac::dts::IL-4) is a good candidate to maintain the anti-inflammatory and proinflammatory balance in the gastrointestinal tract, increasing the levels of IL-10-secreting regulatory cells and, thus, demonstrating the effectiveness of this novel DNA delivery-based strategy.

摘要

背景

炎症性肠病的特征是慢性肠道炎症，可导致肠道黏膜严重受损。因此，了解其病因以及开发新药是治疗此类疾病的重要一步。因此，开发能够递送编码小家鼠白细胞介素4（IL-4）的真核表达载体的乳酸乳球菌菌株，将代表一种制定更有效替代疗法来对抗克罗恩病的新策略。

结果

将小鼠IL-4开放阅读框克隆到真核表达载体pValac::dts中。将所得质粒pValac::dts::IL-4转染到CHO细胞中，以便在体外评估其功能。通过荧光共聚焦显微镜、流式细胞术和酶联免疫吸附测定法观察到，pValac::dts::IL-4转染的细胞产生IL-4，而未转染的细胞和用空载体转染的细胞则不产生。然后，将pValac::dts::IL-4插入乳酸乳球菌MG1363 FnBPA(+)中，以评估重组菌株对三硝基苯磺酸（TNBS）诱导的结肠炎的治疗潜力。胃内给予乳酸乳球菌MG1363 FnBPA(+)（pValac::dts::IL-4）能够减轻结肠炎的严重程度，动物体内IL-12、IL-6水平和髓过氧化物酶（MPO）活性降低；IL-4和IL-10水平升高。最后，对给予TNBS的小鼠分离的肠固有层细胞进行免疫表型分析，以鉴定主要的IL-4和IL-10产生细胞。与其他组相比，给予重组菌株的小鼠中F4/80(+)MHCII(+)Ly6C(-)IL-4(+)、F4/80(+)MHCII(+)Ly6C(-)IL-10(+)、F4/80(+)MHCII(+)Ly6C(-)CD206(+)CD124(+)IL-10(+)和CD4(+)Foxp3(+)IL10(+)细胞的百分比显著更高。