Fasciola hepatica reinfection potentiates a mixed Th1/Th2/Th17/Treg response and correlates with the clinical phenotypes of anemia

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. Methodology/Principal findings 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/Significance 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.

[1]  D. Rollinson,et al.  Outbreak of urogenital schistosomiasis in Corsica (France): an epidemiological case study. , 2016, The Lancet. Infectious diseases.

[2]  G. Weiss.  Anemia of Chronic Disorders: New Diagnostic Tools and New Treatment Strategies. , 2015, Seminars in hematology.

[3]  S. Mas‐Coma,et al.  Diagnosis of human fascioliasis by stool and blood techniques: update for the present global scenario , 2014, Parasitology.

[4]  J. Dalton,et al.  Immunomodulatory molecules of Fasciola hepatica: candidates for both vaccine and immunotherapeutic development. , 2013, Veterinary parasitology.

[5]  Krisztina V. Vukman,et al.  Fasciola hepatica Tegumental Coat Impairs Mast Cells’ Ability To Drive Th1 Immune Responses , 2013, The Journal of Immunology.

[6]  R. Maizels,et al.  Helminth Infections and Host Immune Regulation , 2012, Clinical Microbiology Reviews.

[7]  S. Mas‐Coma,et al.  Field Evaluation of a Coproantigen Detection Test for Fascioliasis Diagnosis and Surveillance in Human Hyperendemic Areas of Andean Countries , 2012, PLoS neglected tropical diseases.

[8]  D. Vignali,et al.  The battle against immunopathology: infectious tolerance mediated by regulatory T cells , 2012, Cellular and Molecular Life Sciences.

[9]  G. Tsokos,et al.  The dysregulation of cytokine networks in systemic lupus erythematosus. , 2011, Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research.

[10]  D. Piedrafita,et al.  Resistance to liver fluke infection in the natural sheep host is correlated with a type‐1 cytokine response , 2011, Parasite immunology.

[11]  J. Aliberti,et al.  Hemophagocytosis causes a consumptive anemia of inflammation , 2011, The Journal of experimental medicine.

[12]  F. Brombacher,et al.  Control of Schistosoma mansoni egg‐induced inflammation by IL‐4‐responsive CD4+CD25−CD103+Foxp3− cells is IL‐10‐dependent , 2010, European journal of immunology.

[13]  V. Kuchroo,et al.  Effector and Regulatory T‐cell Subsets in Autoimmunity and Tissue Inflammation , 2010, Scandinavian journal of immunology.

[14]  Y. Iwakura,et al.  IL-17 Is Necessary for Host Protection against Acute-Phase Trypanosoma cruzi Infection , 2010, The Journal of Immunology.

[15]  G. Mulcahy,et al.  Coordinating innate and adaptive immunity in Fasciola hepatica infection: implications for control. , 2010, Veterinary parasitology.

[16]  A. Espino,et al.  Quantitation of cytokine mRNA by real-time RT-PCR during a vaccination trial in a rabbit model of fascioliasis. , 2010, Veterinary parasitology.

[17]  A. Chauvin,et al.  Immunity against Helminths: Interactions with the Host and the Intercurrent Infections , 2010, Journal of biomedicine & biotechnology.

[18]  P. Brophy,et al.  Major Secretory Antigens of the Helminth Fasciola hepatica Activate a Suppressive Dendritic Cell Phenotype That Attenuates Th17 Cells but Fails To Activate Th2 Immune Responses , 2009, Infection and Immunity.

[19]  G. Mulcahy,et al.  IL‐10 and TGF‐β1 are associated with variations in fluke burdens following experimental fasciolosis in sheep , 2009, Parasite immunology.

[20]  L. Boon,et al.  Infection with a Helminth Parasite Attenuates Autoimmunity through TGF-β-Mediated Suppression of Th17 and Th1 Responses1 , 2009, The Journal of Immunology.

[21]  Y. Belkaid,et al.  Regulatory T cells in the control of host-microorganism interactions (*). , 2009, Annual review of immunology.

[22]  R. Maizels,et al.  Expansion of Foxp3+ Regulatory T Cells in Mice Infected with the Filarial Parasite Brugia malayi1 , 2008, The Journal of Immunology.

[23]  J. Dalton,et al.  Helminth 2‐Cys peroxiredoxin drives Th2 responses through a mechanism involving alternatively activated macrophages , 2008, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[24]  M. Fresno,et al.  Anaemia in advanced chronic fasciolosis. , 2008, Acta tropica.

[25]  Alex Loukas,et al.  Current Status of Vaccines for Schistosomiasis , 2008, Clinical Microbiology Reviews.

[26]  L. Terrazas,et al.  The divergent roles of alternatively activated macrophages in helminthic infections , 2007, Parasite immunology.

[27]  R. Maizels,et al.  CTLA-4 and CD4+CD25+ Regulatory T Cells Inhibit Protective Immunity to Filarial Parasites In Vivo1 , 2007, The Journal of Immunology.

[28]  M. Fresno,et al.  Immune suppression in advanced chronic fascioliasis: an experimental study in a rat model. , 2007, The Journal of infectious diseases.

[29]  R. Maizels.  Regulation of the immune system in metazoan parasite infections. , 2007, Novartis Foundation symposium.

[30]  D. Beiting,et al.  Coordinated Control of Immunity to Muscle Stage Trichinella spiralis by IL-10, Regulatory T Cells, and TGF-β1 , 2007, The Journal of Immunology.

[31]  G. Mulcahy,et al.  Experimental Fasciola hepatica Infection Alters Responses to Tests Used for Diagnosis of Bovine Tuberculosis , 2006, Infection and Immunity.

[32]  J. Kolls,et al.  Regulatory T Cells Dampen Pulmonary Inflammation and Lung Injury in an Animal Model of Pneumocystis Pneumonia1 , 2006, The Journal of Immunology.

[33]  A. Marcilla,et al.  High risk of bacterobilia in advanced experimental chronic fasciolosis. , 2006, Acta tropica.

[34]  Luc Kestens,et al.  Human schistosomiasis , 2006, The Lancet.

[35]  S. O'Neill,et al.  Tissue migration by parasitic helminths - an immunoevasive strategy? , 2005, Trends in parasitology.

[36]  M. Veldhoen,et al.  CD25+ CD4+ T cells compete with naive CD4+ T cells for IL-2 and exploit it for the induction of IL-10 production. , 2005, International immunology.

[37]  T. Mcclanahan,et al.  IL-23 drives a pathogenic T cell population that induces autoimmune inflammation , 2005, The Journal of experimental medicine.

[38]  J. Dalton,et al.  Thioredoxin Peroxidase Secreted by Fasciola hepatica Induces the Alternative Activation of Macrophages , 2005, Infection and Immunity.

[39]  A. O’Garra,et al.  IL-10-producing and naturally occurring CD4+ Tregs: limiting collateral damage. , 2004, The Journal of clinical investigation.

[40]  F. Finkelman,et al.  Interleukin‐4‐ and interleukin‐13‐mediated host protection against intestinal nematode parasites , 2004, Immunological reviews.

[41]  O. Doumbo,et al.  Interleukin‐13 in the skin and interferon‐γ in the liver are key players in immune protection in human schistosomiasis , 2004, Immunological reviews.

[42]  B. Rouse,et al.  CD4+CD25+ Regulatory T Cells Control the Severity of Viral Immunoinflammatory Lesions1 , 2004, The Journal of Immunology.

[43]  Y. Belkaid,et al.  The Pathogenesis of Schistosomiasis Is Controlled by Cooperating IL-10-Producing Innate Effector and Regulatory T Cells , 2004, The Journal of Immunology.

[44]  H. Raadsma,et al.  Immunology of the host-parasite relationship in fasciolosis (Fasciola hepatica and Fasciola gigantica) , 2004 .

[45]  R. Grencis,et al.  Contrasting roles for IL‐10 in protective immunity to different life cycle stages of intestinal nematode parasites , 2003, European journal of immunology.

[46]  E. Moreau,et al.  Modulation of sheep lymphocyte responses by Fasciola hepatica excretory-secretory products. , 2002, Veterinary parasitology.

[47]  C. Boulard,et al.  Early hepatic immune response in rats infected with Fasciola hepatica. , 2002, Veterinary research.

[48]  C. Boulard,et al.  Evaluation of the hepatic NK cell response during the early phase of Fasciola hepatica infection in rats. , 2002, Veterinary research.

[49]  P. Díaz,et al.  Time-course analysis of coproantigens in rats infected and challenged with Fasciola hepatica , 2002, Parasitology Research.

[50]  C. Boulard,et al.  Early hepatic cytokine mRNA expression in experimental rat fasciolosis. , 2002, Veterinary parasitology.

[51]  E. Pearce,et al.  Differential Regulation of Nitric Oxide Synthase-2 and Arginase-1 by Type 1/Type 2 Cytokines In Vivo: Granulomatous Pathology Is Shaped by the Pattern of l-Arginine Metabolism1 , 2001, The Journal of Immunology.

[52]  H. Cejas,et al.  Cytokines involved in the immunosuppressor period in experimental fasciolosis in rats. , 2001, International journal for parasitology.

[53]  S. Mas‐Coma,et al.  Fasciola hepatica and lymnaeid snails occurring at very high altitude in South America , 2001, Parasitology.

[54]  J. Dalton,et al.  Fasciola hepatica cathepsin L cysteine proteinase suppresses Bordetella pertussis‐specific interferon‐γ production in vivo , 2001, Parasite immunology.

[55]  W. F. Gregory,et al.  Identification of tgh-2, a Filarial Nematode Homolog of Caenorhabditis elegans daf-7 and Human Transforming Growth Factor β, Expressed in Microfilarial and Adult Stages of Brugia malayi , 2000, Infection and Immunity.

[56]  M. A. Valero,et al.  Fasciola hepatica: lithogenic capacity in experimentally infested rats and chemical determination of the main stone components , 2000, Parasitology Research.

[57]  C. Boulard,et al.  Local hepatic immune response in rats during primary infection with Fasciola hepatica. , 2000, Parasite.

[58]  J. Dalton,et al.  Fasciola hepatica infection downregulates Th1 responses in mice , 2000, Parasite immunology.

[59]  J. Dalton,et al.  Fasciola hepatica Suppresses a Protective Th1 Response against Bordetella pertussis , 1999, Infection and Immunity.

[60]  J. Dalton,et al.  Immune responses of cattle to experimental anti-Fasciola hepatica vaccines. , 1999, Research in veterinary science.

[61]  R. Panadero,et al.  Subclass profile of specific IgG antibodies in rats challenged during acute and chronic primary infection with Fasciola hepatica , 1999, Parasitology Research.

[62]  J. Cornelissen,et al.  Protection against Fasciola hepatica in the intestine is highly correlated with eosinophil and immunoglobulin G1 responses against newly excysted juveniles , 1999, Parasite immunology.

[63]  S. Goerdt,et al.  Other functions, other genes: alternative activation of antigen-presenting cells. , 1999, Immunity.

[64]  G. Mulcahy,et al.  Lymphocyte and cytokine responses of young cattle during primary infection with Fasciola hepatica. , 1998, Research in veterinary science.

[65]  J. Cornelissen,et al.  Location of induction and expression of protective immunity against Fasciola hepatica at the gut level: a study using an ex vivo infection model with ligated gut segments. , 1998, The Journal of parasitology.

[66]  J. Cornelissen,et al.  Protection to Fasciola hepatica in the gut mucosa of immune rats is associated with infiltrates of eosinophils, IgG1 and IgG2a antibodies around the parasites , 1998, Parasite immunology.

[67]  H. Cejas,et al.  Fasciola hepatica-induced immune suppression of spleen mononuclear cell proliferation: role of nitric oxide. , 1998, Clinical immunology and immunopathology.

[68]  C. Boulard,et al.  Humoral and cellular immune responses to Fasciola hepatica experimental primary and secondary infection in sheep. , 1995, International journal for parasitology.

[69]  M. Toda,et al.  Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. , 1995, Journal of immunology.

[70]  R. Coffman,et al.  Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. , 1994, Immunity.

[71]  A. Bradley,et al.  Multiple defects of immune cell function in mice with disrupted interferon-gamma genes. , 1993, Science.

[72]  A. Trudgett,et al.  Fasciola hepatica in the rat: immune responses associated with the development of resistance to infection , 1992, Parasite immunology.

[73]  S. Yoshihara,et al.  Protection against Fasciola gigantica infection in rats administered metacercarial antigens. , 1985, Research in veterinary science.

[74]  L. Williams,et al.  Cell mediated immunity to liver fluke antigens during experimental Fasciola hepatica infection of cattle , 1985, Parasite immunology.

[75]  C. Chapman,et al.  Fasciola hepatica: comparative studies on fascioliasis in rats and mice. , 1982, International journal for parasitology.

[76]  C. Chapman,et al.  Clonal parasites in the analysis of resistance to reinfection with Fasciola hepatica. , 1981, The American journal of tropical medicine and hygiene.

[77]  G. Mitchell,et al.  Successful passive transfer of resistance to Fasciola hepatica infection in rats by immune serum and transfer factor. , 1981, Research in veterinary science.

[78]  M. Howell,et al.  Age-associated responses in susceptible and resistant rats to infection with Fasciola hepatica. , 1981, International journal for parasitology.

[79]  J. K. Dineen,et al.  The role of the gut in acquired resistance to Fasciola hepatica in the rat. , 1980 .

[80]  D. L. Hughes,et al.  Resistance of the rat to reinfection with Fasciola hepatica and the possible involvement of intestinal eosinophil leucocytes. , 1978, Research in veterinary science.

[81]  D. L. Hughes,et al.  Loss of ability to kill Fasciola hepatica in sensitised rats , 1977, Nature.

[82]  T. J. Hayes,et al.  The early expression of protective immunity to Fasciola hepatica in rats. , 1977, The Journal of parasitology.

[83]  M. Howell,et al.  Fasciola hepatica in rats: effects of age and infective dose. , 1977, International journal for parasitology.

[84]  D. L. Hughes,et al.  The establishment and duration of Fasciola hepatica infections in two strains of rats and the development of acquired resistance. , 1976, Research in veterinary science.

[85]  T. J. Hayes,et al.  Studies on the serum transfer of immunity to Fasciola hepatica in the rat. , 1974, The Journal of parasitology.

[86]  T. J. Hayes,et al.  Serum transfer of immunity to Fasciola hepatica in rats. , 1974, The Journal of parasitology.

[87]  T. J. Hayes,et al.  Acquired immunity and age resistance in rats with chronic fascioliasis. , 1974, The Journal of parasitology.

[88]  F. Ubeira,et al.  Higher physiopathogenicity by Fasciola gigantica than by the genetically close F. hepatica: experimental long-term follow-up of biochemical markers. , 2016, Transactions of the Royal Society of Tropical Medicine and Hygiene.

[89]  S. Mas‐Coma,et al.  Neurological and ocular fascioliasis in humans. , 2014, Advances in parasitology.

[90]  K. P. Murphy,et al.  Janeway's immunobiology , 2007 .

[91]  E. Riley,et al.  Transforming Growth Factor (cid:98) Production Is Inversely Correlated with Severity of Murine Malaria Infection , 1998 .