Rabies virus uniquely reprograms the transcriptome of human monocyte-derived macrophages

Macrophages are amongst the first immune cells that encounter rabies virus (RABV) at virus entry sites. Activation of macrophages is essential for the onset of a potent immune response, but insights into the effects of RABV on macrophage activation are scarce. In this study we performed high-throughput sequencing on RNA extracted from macrophages that were exposed to RABV for 48 hours, and compared their transcriptional profiles to that of non-polarized macrophages (M0), and macrophages polarized towards the canonical M1, M2a and M2c phenotypes. Our analysis revealed that RABV-stimulated macrophages show high expression of several M1, M2a and M2c signature genes. Apart from their partial resemblance to these phenotypes, unbiased clustering analysis revealed that RABV induces a unique and distinct polarization program. Closer examination revealed that RABV induced multiple pathways related to the interferon- and antiviral response, which were not induced under other classical polarization strategies. Surprisingly, our data show that RABV induces an activated rather than a fully suppressed macrophage phenotype, triggering virus-induced activation and polarization. This includes multiple genes with known antiviral (e.g. APOBEC3A, IFIT/OAS/TRIM genes), which may play a role in anti-RABV immunity.</p

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

Carp Il10a and Il10b exert identical biological activities in vitro, but are differentially regulated in vivo

We recently reported on the functional characterization of carp Il10. We showed that carp Il10 is able to downregulate proinflammatory activities by carp phagocytes and promote B cell proliferation, differentiation and antibody production as well as proliferation of memory T cells. Taking advantage of the recent annotation of the carp genome, we completed the sequence of a second il10 paralogue, named il10b, the presence of which was expected owing to the recent (8 million years ago) fourth round of whole genome duplication that occurred in common carp. In the present study we closely compared the two Il10 paralogues and show that Il10a and Il10b have almost identical gene structure, synteny, protein sequence as well as bioactivity on phagocytes. Although the two il10 paralogues show a large overlap in tissue expression, il10b has a low constitutive expression and is highly upregulated upon infection, whereas il10a is higher expressed under basal conditions but its gene expression remains constant during viral and parasitic infections. This differential regulation is most likely due to the observed differences in their promoter regions. Altogether our results demonstrate that gene duplication in carp, although recent, led to sub-functionalization and expression divergence rather than functional redundancy of the Il10 paralogues, yet with very similar protein sequencesAuthors were supported by the European Community's 7th Framework Programme (FP7/2007–2013) and by the 8th (H2020) Framework Programs for Research and Technological Development: MCP under Grant FISHIL10 (PIEF-GA-2011-302444); MF and GFW under Grant TARGETFISH (311993) and IMAQUANIM (FOOD-CT-2005-007103); GFW and ASW were also supported under grant PARAFISHCONTROL (634429). MF was also supported by the Netherlands Organisation for Scientific Research (NWO) under Veni Grant number 11200.Peer reviewe

Cyprinid Herpesvirus 3 Il10 Inhibits Inflammatory Activities of Carp Macrophages and Promotes Proliferation of Igm+ B Cells and Memory T Cells in a Manner Similar to Carp Il10.

Cyprinid herpesvirus 3 (CyHV-3) is the causative agent of a lethal disease of carp and encodes for an Il10 homolog (ORF134). Our previous studies with a recombinant ORF134-deleted strain and the derived revertant strain suggested that cyprinid herpesvirus 3 Il10 (CyHV-3 Il10 [cyhv3Il10]) is not essential for viral replication in vitro, or virulence in vivo. In apparent contrast, cyhv3Il10 is one of the most abundant proteins of the CyHV-3 secretome and is structurally very similar to carp Il10 and also human IL10. To date, studies addressing the biological activity of cyhv3Il10 on cells of its natural host have not been performed. To address the apparent contradiction between the presence of a structurally conserved Il10 homolog in the genome of CyHV-3 and the lack of a clear phenotype in vivo using recombinant cyhv3Il10-deleted viruses, we used an in vitro approach to investigate in detail whether cyhv3Il10 exerts any biological activity on carp cells. In this study, we provide direct evidence that cyhv3Il10 is biologically active and, similarly to carp Il10, signals via a conserved Stat3 pathway modulating immune cells of its natural host, carp. In vitro, cyhv3Il10 deactivates phagocytes with a prominent effect on macrophages, while also promoting proliferation of Igm(+) B cells and memory T cells. Collectively, this study demonstrates a clear biological activity of cyhv3Il10 on cells of its natural host and indicates that cyhv3Il10 is a true viral ortholog of carp Il10. Furthermore, to our knowledge, this is the first report on biological activities of a nonmammalian viral Il10 homolog

Polarization of immune responses in fish : The 'macrophages first' point of view

In this review, we support taking polarized immune responses in teleost fish from a 'macrophage first' point of view, a hypothesis that reverts the dichotomous T helper (TH)1 and TH2 driving forces by building on the idea of conservation of innate immune responses in lower vertebrates. It is plausible that the initial trigger for macrophage polarization into M1 (inflammation) or M2 (healing) could rely only on sensing microbial/parasite infection or other innate danger signals, without the influence of adaptive immunity. Given the long and ongoing debate on the presence/absence of a typical TH1 cytokine environment and, in particular, TH2 cytokine environment in fish immune responses, it stands out that the presence of macrophages with polarized phenotypes, alike M1 and M2, have been relatively easy to demonstrate for fish. We summarize in short present knowledge in teleost fish on those cytokines considered most critical to the dichotomous development of TH1/M1 and TH2/M2 polarization, in particular, but not exclusively, interferon-γ and interleukin (IL)-4/IL-13. We review, in more detail, polarization of fish immune responses taken from the macrophage point of view for which we adopted the simple nomenclature of M1 and M2. We discuss inducible nitric oxide synthase, or NOS-2, as a reliable M1 marker and arginase-2 as a reliable M2 marker for teleost fish and discuss the value of these macrophage markers for the generation of zebrafish reporter lines to study M1/M2 polarization in vivo.</p

The Impact of Culture Variables on a 3D Human In Vitro Bone Remodeling Model:A Design of Experiments Approach

Human in vitro bone remodeling models, using osteoclast–osteoblast cocultures, can facilitate the investigation of human bone remodeling while reducing the need for animal experiments. Although current in vitro osteoclast–osteoblast cocultures have improved the understanding of bone remodeling, it is still unknown which culture conditions support both cell types. Therefore, in vitro bone remodeling models can benefit from a thorough evaluation of the impact of culture variables on bone turnover outcomes, with the aim to reach balanced osteoclast and osteoblast activity, mimicking healthy bone remodeling. Using a resolution III fractional factorial design, the main effects of commonly used culture variables on bone turnover markers in an in vitro human bone remodeling model are identified. This model is able to capture physiological quantitative resorption–formation coupling along all conditions. Culture conditions of two runs show promising results: conditions of one run can be used as a high bone turnover system and conditions of another run as a self-regulating system as the addition of osteoclastic and osteogenic differentiation factors is not required for remodeling. The results generated with this in vitro model allow for better translation between in vitro studies and in vivo studies, toward improved preclinical bone remodeling drug development.</p

Fish Macrophages Show Distinct Metabolic Signatures Upon Polarization

Macrophages play important roles in conditions ranging from host immune defense to tissue regeneration and polarize their functional phenotype accordingly. Next to differences in the use of L-arginine and the production of different cytokines, inflammatory M1 macrophages and anti-inflammatory M2 macrophages are also metabolically distinct. In mammals, M1 macrophages show metabolic reprogramming toward glycolysis, while M2 macrophages rely on oxidative phosphorylation to generate energy. The presence of polarized functional immune phenotypes conserved from mammals to fish led us to hypothesize that a similar metabolic reprogramming in polarized macrophages exists in carp. We studied mitochondrial function of M1 and M2 carp macrophages under basal and stressed conditions to determine oxidative capacity by real-time measurements of oxygen consumption and glycolytic capacity by measuring lactate-based acidification. In M1 macrophages, we found increased nitric oxide production and irg1 expression in addition to altered oxidative phosphorylation and glycolysis. In M2 macrophages, we found increased arginase activity, and both oxidative phosphorylation and glycolysis were similar to control macrophages. These results indicate that M1 and M2 carp macrophages show distinct metabolic signatures and indicate that metabolic reprogramming may occur in carp M1 macrophages. This immunometabolic reprogramming likely supports the inflammatory phenotype of polarized macrophages in teleost fish such as carp, similar to what has been shown in mammals.</p

Fish Macrophages Show Distinct Metabolic Signatures Upon Polarization

Macrophages play important roles in conditions ranging from host immune defense to tissue regeneration and polarize their functional phenotype accordingly. Next to differences in the use of L-arginine and the production of different cytokines, inflammatory M1 macrophages and anti-inflammatory M2 macrophages are also metabolically distinct. In mammals, M1 macrophages show metabolic reprogramming toward glycolysis, while M2 macrophages rely on oxidative phosphorylation to generate energy. The presence of polarized functional immune phenotypes conserved from mammals to fish led us to hypothesize that a similar metabolic reprogramming in polarized macrophages exists in carp. We studied mitochondrial function of M1 and M2 carp macrophages under basal and stressed conditions to determine oxidative capacity by real-time measurements of oxygen consumption and glycolytic capacity by measuring lactate-based acidification. In M1 macrophages, we found increased nitric oxide production and irg1 expression in addition to altered oxidative phosphorylation and glycolysis. In M2 macrophages, we found increased arginase activity, and both oxidative phosphorylation and glycolysis were similar to control macrophages. These results indicate that M1 and M2 carp macrophages show distinct metabolic signatures and indicate that metabolic reprogramming may occur in carp M1 macrophages. This immunometabolic reprogramming likely supports the inflammatory phenotype of polarized macrophages in teleost fish such as carp, similar to what has been shown in mammals.</p

Paralogs of Common Carp Granulocyte Colony-Stimulating Factor (G-CSF) Have Different Functions Regarding Development, Trafficking and Activation of Neutrophils

Mammalian granulocyte colony-stimulating factor (G-CSF; CSF3) is a primary cytokine that promotes the development, mobilization, and activation of neutrophils and their precursors. Teleosts have been reported to possess two paralogs as a likely result of the teleost-wide whole genome duplication (WGD) event, but functional divergence of G-CSF paralogs remains poorly understood. Common carp are an allotetraploid species owing to an additional WGD event in the carp lineage and here, we report on genomic synteny, sequence similarity, and phylogeny of four common carp G-CSF paralogs (g-csfa1 and g-csfa2; g-csfb1 and g-csfb2). G-csfa1 and g-csfa2 show differential and relatively high gene expression levels, while g-csfb1 and g-csfb2 show low basal gene expression levels in most tissues. All paralogs are expressed higher in macrophages than in other leukocyte sub-types and are highly up-regulated by treatment of macrophages with mitogens. Recombinant G-CSFa1 and G-CSFb1 both promoted the proliferation of kidney hematopoietic cells, while only G-CSFb1 induced the differentiation of kidney cells along the neutrophil-lineage. Colony-forming unit assays revealed that G-CSFb1 alone stimulates the formation of CFU-G colonies from head- and trunk-kidney whereas the combination of G-CSFa1 and G-CSFb1 stimulates the formation of both CFU-G and CFU-GM colonies. Recombinant G-CSFa1 and G-CSFb1 also exhibit chemotactic activity against kidney neutrophils and up-regulation of cxcr1 mRNA expression was highest in neutrophils after G-CSFb1 stimulation. Furthermore, G-CSFb1 more than G-CSFa1 induced priming of kidney neutrophils through up-regulation of a NADPH-oxidase component p47 phox . In vivo administration of G-CSF paralogs increased the number of circulating blood neutrophils of carp. Our findings demonstrate that gene duplications in teleosts can lead to functional divergence between paralogs and shed light on the sub-functionalization of G-CSF paralogs in cyprinid fish.</p

DataSheet_1_Rabies virus uniquely reprograms the transcriptome of human monocyte-derived macrophages.docx

Macrophages are amongst the first immune cells that encounter rabies virus (RABV) at virus entry sites. Activation of macrophages is essential for the onset of a potent immune response, but insights into the effects of RABV on macrophage activation are scarce. In this study we performed high-throughput sequencing on RNA extracted from macrophages that were exposed to RABV for 48 hours, and compared their transcriptional profiles to that of non-polarized macrophages (M0), and macrophages polarized towards the canonical M1, M2a and M2c phenotypes. Our analysis revealed that RABV-stimulated macrophages show high expression of several M1, M2a and M2c signature genes. Apart from their partial resemblance to these phenotypes, unbiased clustering analysis revealed that RABV induces a unique and distinct polarization program. Closer examination revealed that RABV induced multiple pathways related to the interferon- and antiviral response, which were not induced under other classical polarization strategies. Surprisingly, our data show that RABV induces an activated rather than a fully suppressed macrophage phenotype, triggering virus-induced activation and polarization. This includes multiple genes with known antiviral (e.g. APOBEC3A, IFIT/OAS/TRIM genes), which may play a role in anti-RABV immunity.</p