Perception, integration and response of the salmonid epithelial cell line RTgill-W1 to infectious and pro-inflammatory agents in vitro

Abstract

The burden of disease is a major issue in aquaculture production. The fish gill is a significant portal of entry for pathogens in fish. To investigate epithelial integrity and innate immune responses in gill epithelia the salmonid gill cell line RTgill-W1 was stimulated with diverse pro-inflammatory and infectious agents. Epithelial integrity in polarised cells expressed as transepithelial electrical resistance (TER) immediately increased after stimulation with different PAMPs (pathogen associated molecular patterns). In parallel tight junction and innate immune gene expression was modulated with bacterial PAMPs, LPS and MDP, internalized through actin dependent and independent endocytic pathways.

The salmonid alphavirus subtype 2 (SAV-2) was used as an infectious agent in RTgill-W1 cells and was found to multiply at a low level. TER was found to be disturbed at an early stage of infection although tight junction related gene expression was not modulated. However, a strong PAMP-driven antiviral response was observed including upregulation of the expression of Rig-like receptor (RLRs) and several interferon stimulated genes (ISGs). Barrier function of trout gill epithelium against infection was also investigated. A bacterial invasion assay using A. salmonicida highlighted the capacity of PAMP pre-treatment to reduce bacterial invasion.

At the level of signal transduction of the phosphoproteome of RTgill-W1 cells in steady state and under poly(I:C) and MDP stimulation was conducted where poly(I:C) stimulated cells presented a higher number of phosphoproteins (360 unique phosphoproteins). These results provide an untargeted view of the key signaling pathways that are rapidly activated in response to PAMP stimulation.

The findings of the present study highlight an integrated response to viral infection and PAMP challenge in fish epithelia that could facilitate the development of novel therapeutic avenues for fish health management in aquaculture.