Modeling Virus-Induced Inflammation in Zebrafish: A Balance Between Infection Control and Excessive Inflammation

Abstract

The inflammatory response to viral infection in humans is a dynamic process with complex cell interactions that are governed by the immune system and influenced by both host and viral factors. Due to this complexity, the relative contributions of the virus and host factors are best studied in vivo using animal models. In this review, we describe how the zebrafish (Danio rerio) has been used as a powerful model to study host-virus interactions and inflammation by combining robust forward and reverse genetic tools with in vivo imaging of transparent embryos and larvae. The innate immune system has an essential role in the initial inflammatory response to viral infection. Focused studies of the innate immune response to viral infection are possible using the zebrafish model as there is a 4-6 week timeframe during development where they have a functional innate immune system dominated by neutrophils and macrophages. During this timeframe, zebrafish lack a functional adaptive immune system, so it is possible to study the innate immune response in isolation. Sequencing of the zebrafish genome has revealed significant genetic conservation with the human genome, and multiple studies have revealed both functional conservation of genes, including those critical to host cell infection and host cell inflammatory response. In addition to studying several fish viruses, zebrafish infection models have been developed for several human viruses, including influenza A, noroviruses, chikungunya, Zika, dengue, herpes simplex virus type 1, Sindbis, and hepatitis C virus. The development of these diverse viral infection models, coupled with the inherent strengths of the zebrafish model, particularly as it relates to our understanding of macrophage and neutrophil biology, offers opportunities for far more intensive studies aimed at understanding conserved host responses to viral infection. In this context, we review aspects relating to the evolution of innate immunity, including the evolution of viral pattern recognition receptors, interferons and interferon receptors, and non-coding RNAs.

Keywords: inflammation; innate immunity; neutrophils; reactive oxidative species; virus infection; zebrafish.

Figure 1

The antiviral response to Influenza A Virus infection. Following IAV entry and infection, single-stranded RNA (ssRNA) and RNA degradation products incorporated into endosomes are recognized by Tlr7 and Tlr8a/b, respectively. In other virus infections, double-stranded RNA by Tlr3 and Tlr22. CpG motifs are recognized and Tlr9 and Tlr21. For Tlr7, Tlr8a/b and Tlr9, the TLR-adaptor, Myd88, activates the NF-κB transcription factor through IkB. NF-κB initiates transcription of inflammatory cytokines, such as Il6, Il1b, and Tnfa. For Tlr3, the TLR-adapter, Ticam1, activates Irf3 that initiates transcription of type I interferons. DAMPs and PAMPs can activate the Nlrp3 inflammasome through activated caspase 1. Activation of RIG-I (Ddx58) by cytosolic viral RNA activates Irf3 and Irf7 transcription factors through Mavs. Irf3 and Irf7 initiate the expression of type 1 interferons that further exacerbates the antiviral innate immune response to infection.

Figure 2

ROS Signaling in Response to Virus Infection. Following infection, production of ROS through the respiratory burst response function to recruit phagocytes (neutrophils and macrophages) to the site of infection and inactivate virus particles. Activation of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (PHOX) complex produces ROS. The PHOX complex is composed to Cyba, Cybb, Ncf1, Ncf2, Ncf4, and Rac1. Activated Nox2 can activate NFκB (p60, p65) that leads to subsequent inflammatory chemokine and cytokine expression. Activated Nox2 can also activate the NRF2 transcription factor through KEAP1 to initiate the expression of antioxidants.

Figure 3

Overlap among miRNA families in zebrafish, mouse, and human genomes. (A) 79 miRNA families are conserved among zebrafish, mouse and human genomes, including miR-142, miR-199 and miR-223. 34 miRNA families are found in the zebrafish, but not in the mouse or human genome. One of the 34 miRNA families is miR-722 which was shown to regulate zebrafish neutrophil migration. 62 miRNA families are found in the mouse, but not in the zebrafish or human genome. 105 miRNA families are found in the human genome, but not in the zebrafish or mouse genome. 83 miRNA families are conserved between the mouse and human genomes that are not found in the zebrafish genome. (B) The origin of the 79 conserved miRNA families are labeled by the last common ancestor for Eumetazoa, Bilateria, Deuterostomia, Chordata, Olfactores, Vertebrata, Osteichthyes, and Gnathostomata with the number of families shown in parentheses. Two of the 79 miRNAs are miR-199 and miR-223 that have roles in neutrophil function. The node of origin for miR-142 and miR-199 is Vertebrata, and Gnathostomata for miR-223.

Figure 4

Immunological Tipping Point in The Inflammatory Response to Virus Infection. A balance between the role of antiviral and hyperinflammatory responses by neutrophils must be maintained to avoid tissue damage during infection by IAV or other viruses. We hypothesize that the modulation of ROS is a central factor in regulating the response to virus infection.