Chitosan reduces the protective effects of IFN-γ2 on grass carp (Ctenopharyngodon idella) against Flavobacterium columnare infection due to excessive inflammation

IFN-gamma is an immunomodulatory factor that has been extensively studied in phenotypes of mammalian macrophages and multifarious inflammatory responses. Usually these studies relied on the classical synergistic activation of IFN-gamma with LPS (LipoPolySaccharides). However, non-mammalian vertebrates, and in particular fish, are not very susceptible to LPS, and easily acquire tolerance upon repeated exposure. Therefore, for studies in fish, it is necessary to replace the classical IFN-gamma+LPS immune system activation method, and find other pathogen-associated molecular patterns (PAMPs) capable of stimulating the fish immune system. Here we used an important farmed fish species, Ctenopharyngodon idella, to study the effects of CiIFN-gamma 2 (C. idella IFN-gamma 2) and chitosan (CS) on its immune responses in vivo and vitro. Our results showed that the combination of CS and CUFN-gamma 2 significantly enhanced the activation of macrophages, with an activation intensity even stronger than in CLIFN-gamma 2 and CiIEN-gamma 2+LPS groups. In vivo, injection of CiIFN-gamma 2 could improve the survival rate of C. idella infected with Flavobacterium columnare, while a combined injection of CUFN-gamma 2+CS only improved protection in the early stages after the challenge. Notably, both injections reduced the bacterial load of viscera and improved the levels of several plasma parameters (TP, T-SOD, LA, and NO). However, a dramatic up-regulation of inflammatory factors, severe inflammatory damage in the intestines and hepatopancreas, and increased mortality in late stages of infection were observed in the CiIFN-gamma 2+CS group. Our findings provide new insights into the macrophage activation phenotypes and inflammatory responses in fish. They also demonstrate that CiIFN-gamma 2 could be used as a potential immunopotentiator, but not in combination with CS. This suggests that selection of immunological adjuvants should be carefully tested experimentally.