The role of SOCS Proteins in salmonid whirling disease

Contents: Whirling disease, caused by the myxosporean parasite Myxobolus cerebralis, afflicts several salmonid species and is considered a serious problem in the fish culture industry and in the natural waters of several countries. Rainbow trout are particularly susceptible and may endure elevated mortality rates, whereas brown trout are known to have a superior resistance to whirling disease. There are differences in disease susceptibility between the two strains of rainbow trout (Oncorhynchus mykiss), the Hofer strain and the Trout lodge strain, and between rainbow trout and brown trout (Salmo trutta). Hypothesis: We believe that the immune response of the latter species stimulates a number of negative feedback regulatory mechanisms to avert excessive immune reactions and subsequent tissue damage. The JAK/STAT pathway is fundamental to cellular signaling and integrates several diverse signaling paradigms in response to extracellular stimuli. The suppressor of cytokine signaling (SOCS) proteins are key in inhibiting cytokine signaling via the JAK/STAT pathway. However, their role in modulating the immune response against whirling disease is completely unknown. Methods: We plan to investigate the significance of SOCS in whirling disease infection and resistance. Several studies suggest that a common link in response to M. cerebralis infection is the involvement of the interferon system. To investigate this point, parasite loads and the expression of SOCS genes in different tissues (caudal fin, skin, muscles,spleen, head kidneyand brain) and in leukocyte populations (B and T cells, myeloid cells) isolated from sampled tissues will be compared at various time points after exposure to the triactinomyxon stages of M. cerebralis, exploring how rainbow trout and brown trout transcriptionally respond to early invasion. Also, morphological changes of M. cerebralis and infiltration of inflammatory cells will be investigated during the development of the parasite in the epidermis. We will investigate the transcriptional response of SOCS genes to infection along with that of several upstream regulators and immune response genes (STAT1, STAT3, IFN-, IFN1, IL6, IL-1ß, IL-10, TGF-ß, arginase-2 and iNOS). New and unique aspect of the project: The project will provide insights into the role of SOCS in regulating the activation and magnitude of innate immunity in rainbow trout (intra-species) and brown trout (inter-species) and will help us to elucidate the mechanisms that underlie the variation in resistance to whirling disease. Key Words: SOCS, JAK/STAT/SOCS pathway, Myxobolus cerebralis, resistant mechanisms, Rainbow trout, Brown trout Running Title: The role of SOCS Proteins in salmonid whirling disease

Whirling disease is a debilitating disease of salmonids caused by the myxozoan parasite Myxobolus cerebralis. There are differences in disease susceptibility between the two strains of rainbow trout (Oncorhynchus mykiss), the Hofer strain (HO) and the Trout lodge (TL) strain, and between rainbow trout and brown trout (Salmo trutta), the original host of M. cerebralis. The aim of the proposed study is to explore the role of the suppressor of cytokine signaling (SOCS) proteins SOCS1 and SOCS3, in the varying susceptibility to whirling disease infection exhibited by rainbow trout and brown trout. Fish were sampled to assess the transcriptional response of SOCS genes to M. cerebralis infection, along with that of several upstream regulators and immune response genes in different tissues (caudal fin (CF), spleen (SP) and head kidney (HK)) and the leukocyte populations (B-and T-cells, myeloid cells). For the first time, a flow cytometry analysis was performed with leukocytes extracted from the fins of the fish, allowing the proper analysis of the cell composition under a parasitic infection. The results show that M. cerebralis stimulates leukocytes trafficking in rainbow trout. A local inflammatory response mediated by myeloid cells in TL was observed correlating to parasite load quantified in CF. In HO, T lymphocytes likely drive the immune response and mediate resistance against M. cerebralis. Resistant fish demonstrate a more moderate, putative T lymphocyte-mediated immune response, which may contribute to their resistance. M. cerebralis induced the expression of SOCS1, the IL-6-dependent SOCS3, the anti- inflammatory cytokine IL-10 and the Treg associated transcription factor FOXP3 in TL fish at multiple time points, which likely caused a restricted STAT1 and STAT3 activity affecting the Th17/Treg17 balance. Conversely, in HO fish, the expression of SOCS1 and SOCS3 was restrained, whereas the expression of STAT1 and IL-23-mediated STAT3 was induced potentially enabling more controlled immune responses, accelerating parasite clearance and elevating resistance. Similar to HO, brown trout fish reduced the parasite load and demonstrated effective immune responses, likely by keeping pro-inflammatory and anti- inflammatory cytokines in balance whilst stimulating efficient Th17-mediated immunity. This project increases knowledge on salmonids immune response to M. cerebralis and helps us to understand the underlying mechanisms of WD resistance.