title: 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.
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.
authors: Laboratório de Tecnologia Genética, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors: Bianca Mendes
authors: BM
authors: Souza
authors: Laboratório de Tecnologia Genética, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors: Tatiane Melo
authors: TM
authors: Preisser
authors: Laboratório de Tecnologia Genética, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors: Vanessa Bastos
authors: VB
authors: Pereira
authors: Laboratório de Tecnologia Genética, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors: Meritxell
authors: M
authors: Zurita-Turk
authors: Laboratório de Tecnologia Genética, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors: Camila Prósperi
authors: CP
authors: de Castro
authors: Laboratório de Tecnologia Genética, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors: Vanessa Pecini
authors: VP
authors: da Cunha
authors: Departamento de Ciências Biológicas, Instituto Federal do Paraná, Foz do Iguaçu, Paraná, Brazil.
authors: Rafael Pires
authors: RP
authors: de Oliveira
authors: Laboratório de Imunobiologia, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
authors: Ana Cristina
authors: AC
authors: Gomes-Santos
authors: Laboratório de Imunobiologia, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
authors: Ana Maria Caetano
authors: AM
authors: de Faria
authors: Laboratório de Alergia e Inflamação, Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors: Denise Carmona Cara
authors: DC
authors: Machado
authors: INRA, UMR1319 Micalis, Jouy-en-Josas, France.
authors: Jean-Marc
authors: JM
authors: Chatel
authors: Laboratório de Genética Celular e Molecular Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors: Vasco Ariston de Carvalho
authors: VA
authors: Azevedo
authors: INRA, UMR1319 Micalis, Jouy-en-Josas, France.
authors: Philippe
authors: P
authors: Langella
authors: Laboratório de Tecnologia Genética, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. miyoshi@icb.ufmg.br.
authors: Anderson
authors: http://orcid.org/0000-0002-0480-8993
authors: A
authors: Miyoshi
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D016207:Cytokines / Q000276:immunology / Q000378:metabolism; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D007249:Inflammation / Q000139:chemically induced / Q000628:therapy*; D015212:Inflammatory Bowel Diseases / Q000276:immunology / Q000517:prevention & control / Q000628:therapy*; D016753:Interleukin-10 / Q000378:metabolism*; D015847:Interleukin-4 / Q000235:genetics* / Q000276:immunology / Q000627:therapeutic use; D007413:Intestinal Mucosa / Q000166:cytology / Q000276:immunology; D013294:Lactococcus lactis / Q000235:genetics*; D051379:Mice; D009092:Mucous Membrane / Q000276:immunology / Q000648:ultrastructure; D014162:Transfection
chemical_list: D016207:Cytokines; C556062:IL10 protein, mouse; D016753:Interleukin-10; D015847:Interleukin-4; D004247:DNA
keywords: Crohn’s disease; Interleukin 10; Interleukin 4; Lactococcus lactis; Regulatory cells