Immunological stimuli change expression of genes and neutrophil function in fathead minnow Pimephales promelas Rafinesque

Abstract

Fathead minnows Pimephales promelas were exposed to lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid [poly(I:C)] to observe immunological responses during simulated bacterial and viral challenge at the level of gene expression and granulocyte function. Complementary DNA libraries were created from LPS- and poly(I:C)-treated fish and c. 5000 expressed sequence tags (ESTs) were sequenced. The ESTs were subjected to BLASTx analysis and 1500 genes were annotated, grouped by function and 20 immune genes were selected for expression studies by real-time PCR. Lipopolysaccharide treatment significantly downregulated expression of interferon regulatory factor 2 binding protein 1 (nine-fold), Chemokine (C-X-C motif) ligand 12a (three-fold) and TNF-related apoptosis-inducing ligand, TRAIL (two-fold). In poly(I:C)-treated fish, a significant upregulation was observed for IFN-inducible and antiviral proteins belonging to the family of Mx proteins (73-fold) and chemokine CCL-C5a (28-fold). Blood neutrophil count was significantly increased in poly(I:C)-treated fish at 24 and 48 h post-injection. Neutrophil extracellular trap release and respiratory burst of kidney granulocytes were suppressed in poly(I:C)-treated fish, while degranulation of primary granules was not affected significantly by the treatment. The changes in gene expression and neutrophil function in P. promelas exposed to LPS and poly(I:C) support the use of this species as an alternative model for studies of pathogen effects on the innate immune system of fishes.