Polarized innate immunity: conservation of macrophage polarization in carp

Rising demands for animal protein have caused an increase and further intensification of aquaculture over the last decades, which has subsequently led to increased disease pressure. Next to preventative vaccines as a solution, there is a drive to explore preventative approaches based on immunomodulation of innate immune responses. In either case, it isclear that a more detailed knowledge of innate immune responses is essential to help combat infectious diseases in aquaculture. Therefore, the overall aim of this thesis is to provide fundamental knowledge of the fish’ innate immune system and characterize polarized innate immune responses in carp with the emphasis on macrophages.In chapter 1 we touch upon the relevance of carp as an aquaculture species and explain why macrophages are considered essential players in innate immune responses, particularly in lower and cold-blooded vertebrates such as fish. We introduce macrophages as highly plastic cell types, introduce their activation signals and introduce the concept of macrophage polarization as it has been defined for mammalian macrophages. Then, following discussion of the framework that will help define macrophage polarization, we briefly summarize existing indications for the presence of polarized macrophages in carp. Finally, we shortly discuss how our findings can aid the development of immunomodulators that could help improve fish health in the context of aquaculture.We start by thoroughly reviewing the existing literature on macrophage polarization in fish in chapter 2. We review the stimuli frequently used to polarize macrophages in mammals, and the conservation of cytokines often associated with T helper 1 and T helper 2 subsets. We discuss approaching macrophage polarization in fish from a ‘macrophages first’ point of view and consider the plausibility that polarization in fish macrophages could rely primarily on sensing microbial infection or other innate danger signals. Furthermore, we discuss preliminary but promising markers to read out M1 and M2 macrophage responses in fish, including inos as a conserved marker for M1 and arginase 2 as a marker for M2 fish macrophages.Based on evidence that several key functions of mammalian macrophages are also conserved in fish, we describe comprehensive functional and transcriptional phenotypes of polarized carp macrophages in chapter 3. We combine information on established nitric oxide (NO) and arginase assays with morphological differences to first, confirm M1 and M2 macrophage polarization in carp and second, use a sequencing approach to elucidate transcriptional profiles of these M1 and M2 macrophages. We confirm that carp macrophages can polarize into M1- and M2 phenotypes with conserved functions and with transcriptional profiles corresponding to mammalian macrophages. Carp M1 macrophages produce NO and increase expression of pro-inflammatory genes including il6, il12 and saa. Carp M2 macrophages show increased arginase activity and increase expression of anti-inflammatory mediators, including cyr61, timp2b and tgm2b. Furthermore, we list several candidate markers that can help discriminate between M1 and M2 macrophages of teleost fish. Finally, we touch upon the importance of our findings for the identification of gene targets to generate new transgenic zebrafish for detailed and in vivo studies on M1 and M2 macrophages. Above all, we discuss the striking degree of evolutionary conservation of macrophage polarization in a lower vertebrate.In chapter 4 we study how M1 macrophages polarized with LPS contribute to neutrophil responses as the major producers of granulocyte colony stimulating factor (Csf3/G-csf). We identify four carp Csf3 paralogs and study basal expression patters in different organs and cell-types. We pinpoint M1 macrophages as the major producers of Csf3 and show that expression is highly increased upon stimulation with mitogens. We characterize both Csf3a and Csf3b as promotors of proliferation in kidney hematopoietic cells, while Csf3b in particular induces neutrophil differentiation. Both CSF3 paralogs were chemotactic for neutrophils and both Csf3a and Csf3b enhance the respiratory burst capacity of neutrophils. The results indicate that M1 macrophage polarization potentiates the proliferation, activation and inflammatory function of neutrophils.In mammals, M1 macrophages show metabolic reprogramming toward glycolysis, while M2 macrophages rely on oxidative phosphorylation to generate energy. In chapter 5, we hypothesize that functional conservation of polarized macrophages in carp could also indicate conservation of associated energy metabolism. Therefore, we study the metabolic profiles of carp macrophages polarized towards M1 and M2 extremes. Using real-time extracellular flux analysis (Seahorse) we determine oxidative phosphorylation and glycolysis in M1 and M2 macrophages. Like mammalian M1 macrophages, we found upregulation of irg1 and altered oxidative phosphorylation and glycolysis in carp M1 macrophages. In carp M2 macrophages both oxidative phosphorylation and glycolysis were similar to controls. The changes in metabolism of M1 macrophages indicated that metabolic reprogramming may occur in carp M1 macrophages, resulting in distinct metabolic signatures in M1 and M2 carp macrophages. The immunometabolic reprogramming of M1 macrophages likely supports the inflammatory phenotype of these cells in teleost fish such as carp, similar to what has been shown in mammals.In the General discussion (chapter 6) we discuss our findings primarily in the light of evolutionary conservation of macrophage polarization. We discuss the current knowledge on conservation and use of polarizing cytokines to direct carp macrophages as a next step to expand and refine our understanding of carp macrophage responses. We apply our proposed M1 and M2 markers to elucidate a ‘common inflammatory’ phenotype and present preliminary results that may indicate that, similar to mammals, the M2 phenotype in fish could include more than just the ‘traditional’ M2(a) subset. Next, we scrutinize the use of the candidate markers identified in chapter 3 for analysis in tissue collected from in vivo infection studies. We discuss an integrated approach to immunology and metabolism and the potential of immunometabolism as new read-out system for polarized innate immune responses in lower vertebrates. Finally, we discuss the plasticity of fish macrophages and support the adoption of a ‘spectrum view’ on polarization of fish macrophages