title: Fasciola hepatica reinfection potentiates a mixed Th1/Th2/Th17/Treg response and correlates with the clinical phenotypes of anemia.
abstract: BACKGROUND
  Fascioliasis is a severe zoonotic disease of worldwide extension caused by liver flukes. In human fascioliasis hyperendemic areas, reinfection and chronicity are the norm and anemia is the main sign. Herein, the profile of the Th1/Th2/Th17/Treg expression levels is analyzed after reinfection, correlating them with their corresponding hematological biomarkers of morbidity.
  
  
  RESULTS
  The experimental design reproduces the usual reinfection/chronicity conditions in human fascioliasis endemic areas and included Fasciola hepatica primo-infected Wistar rats (PI) and rats reinfected at 8 weeks (R8), and at 12 weeks (R12), and negative control rats. In a cross-sectional study, the expression of the genes associated with Th1 (Ifng, Il12a, Il12b, Nos2), Th2 (Il4, Arg1), Treg (Foxp3, Il10, Tgfb, Ebi3), and Th17 (Il17) in the spleen and thymus was analyzed. After 20 weeks of primary infection, PI did not present significant changes in the expression of those genes when compared to non-infected rats (NI), but an increase of Il4, Arg1 and Ifng mRNA in the spleen was observed in R12, suggesting the existence of an active mixed Th1/Th2 systemic immune response in reinfection. Foxp3, Il10, Tgfb and Ebi3 levels increased in the spleen in R12 when compared to NI and PI, indicating that the Treg gene expression levels are potentiated in chronic phase reinfection. Il17 gene expression levels in R12 in the spleen increased when compared to NI, PI and R8. Gene expression levels of Il10 in the thymus increased when compared to NI and PI in R12. Ifng expression levels in the thymus increased in all reinfected rats, but not in PI. The clinical phenotype was determined by the fluke burden, the rat body weight and the hemogram. Multivariate mathematical models were built to describe the Th1/Th2/Th17/Treg expression levels and the clinical phenotype. In reinfection, two phenotypic patterns were detected: i) one which includes only increased splenic Ifng expression levels but no Treg expression, correlating with severe anemia; ii) another which includes increased splenic Ifng and Treg expression levels, correlating with a less severe anemia.
  
  
  CONCLUSIONS
  In animals with established F. hepatica infection a huge increase in the immune response occurs, being a mixed Th2/Treg associated gene expression together with an expression of Ifng. Interestingly, a Th17 associated gene expression is also observed. Reinfection in the chronic phase is able to activate a mixed immune response (Th1/Th2/Th17/Treg) against F. hepatica but T and B proliferation to mitogens is strongly suppressed in all infected rats vs control in the advanced chronic phase independently of reinfection The systemic immune response is different in each group, suggesting that suppression is mediated by different mechanisms in each case. Immune suppression could be due to the parasite in PI and R8 rats and the induction of suppressive cells such as Treg in R12. This is the first study to provide fundamental insight into the immune profile in fascioliasis reinfection and its relation with the clinical phenotypes of anemia.
authors.affiliation: Departamento de Parasitología, Facultad de Farmacia, Universidad de Valencia, Av. Vicent Andrés Estellés s/n, Burjassot, Valencia, Spain.
authors.forename: M Adela
authors.identifier: http://orcid.org/0000-0003-1584-8330
authors.initials: MA
authors.lastname: Valero
authors.affiliation: Departamento de Parasitología, Facultad de Farmacia, Universidad de Valencia, Av. Vicent Andrés Estellés s/n, Burjassot, Valencia, Spain.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Perez-Crespo
authors.affiliation: Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), C/ Nicolás Cabrera 1, Cantoblanco, Madrid, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Chillón-Marinas
authors.affiliation: Departamento de Parasitología, Facultad de Farmacia, Universidad de Valencia, Av. Vicent Andrés Estellés s/n, Burjassot, Valencia, Spain.
authors.forename: Messaoud
authors.initials: M
authors.lastname: Khoubbane
authors.affiliation: Departamento de Parasitología, Facultad de Farmacia, Universidad de Valencia, Av. Vicent Andrés Estellés s/n, Burjassot, Valencia, Spain.
authors.forename: Carla
authors.initials: C
authors.lastname: Quesada
authors.affiliation: Departamento de Parasitología, Facultad de Farmacia, Universidad de Valencia, Av. Vicent Andrés Estellés s/n, Burjassot, Valencia, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Reguera-Gomez
authors.affiliation: Departamento de Parasitología, Facultad de Farmacia, Universidad de Valencia, Av. Vicent Andrés Estellés s/n, Burjassot, Valencia, Spain.
authors.forename: Santiago
authors.initials: S
authors.lastname: Mas-Coma
authors.affiliation: Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), C/ Nicolás Cabrera 1, Cantoblanco, Madrid, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Fresno
authors.affiliation: Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), C/ Nicolás Cabrera 1, Cantoblanco, Madrid, Spain.
authors.forename: Núria
authors.initials: N
authors.lastname: Gironès
mesh_terms: D000740:Anemia / Q000276:immunology*; D000818:Animals; D049109:Cell Proliferation / Q000235:genetics / Q000502:physiology; D003430:Cross-Sectional Studies; D005210:Fasciola hepatica / Q000276:immunology* / Q000472:pathogenicity*; D051858:Forkhead Transcription Factors / Q000378:metabolism; D016753:Interleukin-10 / Q000378:metabolism; D007378:Interleukins / Q000378:metabolism; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012908:Snails / Q000469:parasitology; D018417:Th1 Cells / Q000378:metabolism*; D058504:Th17 Cells / Q000378:metabolism*; D018418:Th2 Cells / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
chemical_list: D051858:Forkhead Transcription Factors; D007378:Interleukins; D053773:Transforming Growth Factor beta1; D016753:Interleukin-10