(C,D) PEC cells were stained for F4/80, CD11b, MHCII, CD206, and PD-L2 and analyzed by flow cytometry. but reduced TNF production upon re-stimulation with FHTE or TLR ligands and this was reversed by inhibitors of DNA methylation. In contrast, macrophages trained with -glucan or Bacillus CalmetteCGurin had enhanced TNF production upon re-stimulation with Pam3cys or LPS. Furthermore, FHTE-trained macrophages had enhanced expression of markers of alternative activated macrophages (AAM). Macrophages from mice treated with FHTE expressed markers of AAM and had heightened IL-10 and IL-1RA production in response to FHTE or TLR ligands and had suppressed TNF and IL-12p40 production. Macrophages from mice treated with FHTE had reduced APC function and inhibited IL-17 production and the encephalitogenic activity of T cells in the experimental autoimmune encephalomyelitis (EAE) model. In addition, mice pre-treated with FHTE were resistant to induction of EAE and this was associated with a significant reduction in IL-17-producing and CD4 T cells infiltrating the CNS. Our findings reveal that cells of the innate immune system can be trained or to be more anti-inflammatory by exposure to helminth products and this protects mice against the induction of a T cell-mediated autoimmune disease. provoke anti-inflammatory immune response (9, 10, 25), we reasoned that may be a useful source of products for inducing anti-inflammatory trained immunity. Our findings demonstrate that total extract (FHTE) can train macrophages and to be more anti-inflammatory, suppressing effector Th1 and Th17 responses. Furthermore, mice pre-treated with two single injections of FHTE were resistant to the development of experimental autoimmune encephalomyelitis (EAE) and this was mediated by suppression of pathogenic T cell responses in the periphery and reduced infiltration of Aclidinium Bromide encephalitogenic T cells into the CNS. Materials and Methods Mice C57BL/6 mice were bred in house from established colonies. All mice were maintained according to European Union regulations, and experiments were performed under license (AE19136/P042) from the Irish Health Products Regulation Authority with Aclidinium Bromide approval from the Trinity College Dublin BioResources Ethics Committee. All mice were housed under specific pathogen-free conditions. All mice within experiments were age and sex matched. Preparation of FHTE Aclidinium Bromide Adult flukes were collected from infected bovine livers at a local abattoir (Kildare Chilling Ltd). Freshly isolated flukes were washed several times in PBS containing 100 g/ml Penicillin-Streptomycin (PS, Sigma) to remove contaminants and cellular debris and transported to the PTGIS lab. Live flukes were incubated at 5C6 worms per 3 ml in PBS/PS overnight in a cell culture incubator at 37C and 5% CO2. Supernatants were removed, and the flukes were washed three times in PBS/PS before being washed twice with PBS. Supernatants were decanted after the last wash and flukes were mechanically homogenized for 5 min. The homogenate was centrifuged for 5 min at 2,000 g to remove large debris followed by centrifugation for 30 min at 15,000 g. The total soluble fraction (FHTE) was filtered through a 5 mm filter and then a 0.2 m filter. The sterile homogenate was harvested, aliquoted and stored at ?80C. The concentration of FHTE used was based on protein content determined by the bicinchoninic acid assay. For studies, FHTE was used at a concentration of either 1.25% v/v (130 g /ml) or 2.5% v/v (260 g /ml). For studies, each mouse was injected with 50 g of FHTE in 200 l (250 g/ml) of PBS. Generation of Bone Marrow-Derived Macrophages (BMDMs) BMDMs were generated from C57BL/6 mice. Bone marrow was flushed from the bones using a 25G needle attached to a 20 ml syringe containing RPMI medium and cell clusters were disrupted by aspirating the cell suspension through a 19G needle. The single cell suspension was centrifuged at 300 g for 5 min before being resuspended in 2 ml of ammonium chloride lysis solution for 2 min in order to lyse the red blood cells. Cells were washed in RPMI and centrifuged at 300 g for 5 min and cultured in petri dishes at 1 106 cells/ml in RPMI supplemented with 20% v/v of macrophage-colony stimulating factor (M-CSF) in the form of supernatants obtained from culture of the L929 cell line. M-CSF-producing L929 cells were obtained by transfection having a plasmid encoding murine M-CSF. On day time 3, additional 2 ml of L929 medium was added to each.