bioRxiv preprint doi: https://doi.org/10.1101/2021.06.21.449266; this version posted June 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Timing of transcriptomic peripheral blood mononuclear cell responses 2 of sheep to Fasciola hepatica infection differs from those of cattle, 3 reflecting different disease phenotypes 4 Dagmara A. Niedziela1, Amalia Naranjo-Lucena1, Verónica Molina-Hernández2, John A. 5 Browne3, Álvaro Martínez-Moreno4, José Pérez2, David E. MacHugh3,5, Grace Mulcahy1,5* 6 1 UCD School of Veterinary Medicine, University College Dublin, Dublin, Ireland 7 2 Departamento de Anatomía y Anatomía Patológica Comparadas y Toxicología, Facultad de 8 Veterinaria, Universidad de Córdoba, Spain 9 3 Animal Genomics Laboratory, UCD School of Agriculture and Food Science, University 10 College Dublin, Dublin, Ireland 11 4 Departamento de Sanidad Animal (Parasitología), Facultad de Veterinaria, Universidad de 12 Córdoba, Spain 13 5 UCD Conway Institute of Biomolecular and Biomedical Research, University College 14 Dublin, Dublin, Ireland 15 16 * Correspondence: 17 Grace Mulcahy 18 [email protected] 19 20 Keywords: transcriptomics, sheep, PBMC, Fasciola, anti-inflammatory response, response to 21 short RNAs 22 23 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.21.449266; this version posted June 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 24 Abstract 25 Infection with the zoonotic trematode Fasciola hepatica, common in many regions with a 26 temperate climate, leads to delayed growth and loss of productivity in cattle, while infection in 27 sheep can have more severe effects, potentially leading to death. Previous transcriptomic 28 analyses revealed upregulation of TGFB1, cell death and Toll-like receptor signalling, T-cell 29 activation, and inhibition of nitric oxide production in macrophages in response to infection. 30 However, the differences between ovine and bovine responses have not yet been explored. The 31 objective of this study was to further investigate the transcriptomic response of ovine peripheral 32 blood mononuclear cells (PBMC) to F. hepatica infection, and to elucidate the differences 33 between ovine and bovine PBMC responses. 34 Sixteen male Merino sheep were randomly assigned to infected or control groups (n = 8 per 35 group) and orally infected with 120 F. hepatica metacercariae. Transcriptomic data was 36 generated from PBMC at 0, 2 and 16 weeks post-infection (wpi), and analysed for differentially 37 expressed (DE) genes between infected and control animals at each time point (analysis 1), and 38 for each group relative to time 0 (analysis 2). Analysis 2 was then compared to a similar study 39 performed previously on bovine PBMC. 40 A total of 453 DE genes were found at 2 wpi, and 2 DE genes at 16 wpi (FDR < 0.1, analysis 41 1). Significantly overrepresented biological pathways at 2 wpi included role of PKR in 42 interferon induction and anti-viral response, death receptor signalling and RIG-I-like receptor 43 signalling, which suggested that an activation of innate response to intracellular nucleic acids 44 and inhibition of cellular apoptosis were taking place. Comparison of analysis 2 with the 45 previous bovine transcriptomic study revealed that anti-inflammatory response pathways 46 which were significantly overrepresented in the acute phase in cattle, including IL-10 47 signalling, Th2 pathway, and Th1 and Th2 activation were upregulated only in the chronic 48 phase in sheep. We propose that the earlier activation of anti-inflammatory responses in cattle, 49 as compared with sheep, may be related to the general absence of acute clinical signs in cattle. 50 These findings offer scope for “smart vaccination” strategies for this important livestock 51 parasite. 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.21.449266; this version posted June 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 52 1 Introduction 53 Fasciola hepatica is a zoonotic trematode that affects livestock (sheep, cattle, goats) as well as 54 humans. Human cases of fasciolosis occur mainly in Latin America, Africa and Asia, with 55 approximately 2.5 million people affected worldwide (Mas-Coma et al., 1999, WHO, 2006). 56 Sporadic human cases also occur in Europe as a result of the consumption of raw vegetables 57 such as parsley or watercress, grown on irrigated or flooded land (Rondelaud et al., 2000). 58 F. hepatica infection is an important animal health issue in ruminants in endemic areas 59 (Torgerson and Claxton, 1999). In Ireland, F. hepatica seroprevalence is reported as 75.4% to 60 82% of dairy herds, and within-herd prevalence is estimated at ≤ 50% (Bloemhoff et al., 2015, 61 Selemetas et al., 2015). In Northern Ireland, 63.15% cattle herds were positive for F. hepatica, 62 with an overall animal level prevalence of 23.68% (Byrne et al., 2016). Horses can also become 63 infected, with a 9.5% prevalence of F. hepatica antibodies reported in a post-mortem survey 64 of horses in Ireland (Quigley et al., 2017). The cost of livestock infection includes productivity 65 losses (e.g., meat, milk, wool) as well as reduced fertility and condemnation of livers as unfit 66 for human consumption (Hurtrez-Boussès et al., 2001, Schweizer et al., 2005). While F. 67 hepatica infection in cattle leads to delayed growth and loss of productivity, acute infection in 68 sheep can have more severe effects. The severe disease presentation in sheep is linked to a 69 rapid acute response and can lead to sudden death (Behm and Sangster, 1999). Anthelmintic 70 resistance in F. hepatica populations is an increasingly important issue, with resistance to 71 triclabendazole of particular concern (Martínez-Valladares et al., 2014, Kelley et al., 2016), as 72 this is the only drug effective against early immature flukes. Hence, there has been significant 73 attention given to developing a vaccine effective in protecting ruminants against fasciolosis. 74 While promising vaccine trials have been conducted, repeatability and consistency has been an 75 issue (Molina-Hernández et al., 2015, McManus, 2020). 76 The ruminant immune response to F. hepatica infection involves an increase in peripheral 77 eosinophil numbers, alternative activation of macrophages, and suppression of Th1 78 responsiveness to the parasites themselves and to bystander antigens (Flynn et al., 2010, O'Neill 79 et al., 2001). The transcriptomic response of peripheral blood mononuclear cells (PBMC) to F. 80 hepatica in cattle has been characterised by apoptosis of antigen-presenting cells (APCs), liver 81 fibrosis and a Th2 response, with TNF, IL1B, and DUSP1, APP, STAT3 and mir-155 as 82 important upstream regulator genes leading to hepatic fibrosis and apoptosis of APCs or 83 migration and chemotaxis of leukocytes (Garcia-Campos et al., 2019). A balanced Treg-Th2 84 response was present in the acute phase with increased polarization towards a Th2 response 85 during the chronic phase of infection. 86 The transcriptomic response of ovine PBMC has also been characterised previously. The 87 response at 2 and 8 weeks post-infection (wpi) was associated with upregulation of the TGF-β 88 signalling pathway, the complement system, chemokine signalling and T-cell activation. 89 Groups of genes were identified which were important for the immune response to the parasite 90 and included those coding for lectins, pro-inflammatory molecules from the S100 family and 91 transmembrane glycoproteins from the CD300 family. Early infection was characterised by 92 positive activation of T-cell migration and leukocyte activation, while the in the later stage 93 lipoxin metabolic processes and Fc gamma R-mediated phagocytosis were among the 94 upregulated pathways (Alvarez Rojas et al., 2016). Another study which looked at the ovine 95 acute and chronic response at 1 and 14 wpi identified upregulation of TGFβ signalling, 96 production of nitric oxide in macrophages, death receptor signalling and IL-17 signalling at 2 97 wpi, as well as Toll-like receptor (TLR) and p53 signalling in both the acute and chronic phase 98 (Fu et al., 2016). Overall, these studies suggest a drive towards an anti-inflammatory response 99 and tissue repair in F. hepatica infection in both sheep and cattle. 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.21.449266; this version posted June 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 100 The objective of this study was to further investigate the pathways involved in the response of 101 ovine PBMC to F. hepatica infection, and to compare those with bovine responses to aid in 102 developing effective vaccine strategies. 103 104 2 Materials and methods 105 2.1 Animal trial outline 106 The animal trial has been described previously by Naranjo-Lucena et al. (2021). Briefly, 107 sixteen male Merino sheep were obtained from a liver fluke-free farm at 9 months of age. All 108 animals were tested monthly for parasite eggs by faecal sedimentation. Prior to challenge, all 109 animals were tested for serum IgG specific for FhCL1 by an enzyme-linked immunosorbent 110 assay (ELISA).