The nucleotide-binding domain, leucine-rich repeat containing (NLR) protein family is essential for regulating the innate immune response. NLR proteins are a family of pattern-recognition sensors that are activated by cytosolic pathogen-associated molecular patterns and damage-associated molecular patterns. Following activation, NLR proteins initiate proinflammatory signaling cascades or restrain overzealous immune responses (Davis et al., 2011; Lich and Ting, 2007). In humans, dysfunction of NLR proteins has been linked to an assortment of inflammatory diseases. For example, mutations in the prototypical NLR proteins nucleotide-binding oligomerization domain-containing 1 (NOD1) and NOD2 have been associated with human inflammatory conditions, including asthma, atopy and inflammatory bowel diseases (IBDs) (Hugot et al., 2001; Ogura et al., 2001) (reviewed in Elinav et al., 2011). NOD1 and NOD2 sense muropeptides and muramyl dipeptide, respectively, which are components of bacterial peptidoglycan. Additionally, NOD2 has been shown to sense single-stranded RNA and N-glycol muramyl dipeptide, which indicates that this receptor might also participate in initiating the immune response to viruses or mycobacteria (Coulombe et al., 2009; Sabbah et al., 2009). Following their activation, NOD1 and NOD2 initiate signaling cascades that result in the activation of both nuclear factor-κB (NFκB) and mitogen-activated protein kinase (MAPK) pathways.
Despite the wealth of data associated with human and mouse NLR protein family members, many aspects of the signaling pathways that they initiate and their roles in vivo are still elusive. Traditionally, mouse models have been used as the model organism of choice to study the in vivo relevance of NLR proteins. However, a paper in this issue of Disease Models & Mechanisms demonstrates that zebrafish can also be a useful model for studying the role of these innate immune sensors (Oehlers et al., 2011).
Over the last 20 years, the zebrafish has risen to prominence as a bona fide research model and has proven to be an excellent vertebrate system for immunology studies. Historically, zebrafish models provided a powerful system to conduct forward genetic screens, which allowed a tremendous amount of progress to be made in understanding organogenesis and development. Zebrafish are also amenable to reverse genetic studies. Gene expression can be effectively knocked down in zebrafish using morpholinos (modified antisense DNA oligonucleotides). In addition to the powerful tools available for genetic manipulation, the small size (<5 cm) and prolific reproduction (200–300 progeny/pair/week) make zebrafish highly cost effective and uniquely suited for high-throughput in vivo screening (reviewed in Meeker and Trede, 2008). In immune system studies, the optical transparency of zebrafish during early development has allowed researchers to generate an impressive amount of in situ and real-time data regarding leukocyte trafficking and immune system development. Although zebrafish offer several advantages over mammalian models, there are also several key disadvantages that have restricted their use in immunology research: there is a lack of antibodies available for zebrafish proteins, immunology-focused cell culture techniques are underdeveloped and the techniques to generate conventional knockout organisms have yet to be developed (Meeker and Trede, 2008).
The new study by Oehlers et al. used zebrafish as a model system to provide insight into the contribution of NLR protein family members to IBD pathogenesis (Oehlers et al., 2011). IBDs are a group of disorders that affect the gastrointestinal system and are widely prevalent in human populations. The most common IBDs diagnosed are Crohn’s disease and ulcerative colitis. Both of these diseases have strong genetic and environmental factors that result in a dysfunctional gastrointestinal immune response to the gut microbiota. In the case of Crohn’s disease, at least 12 susceptibility genes have been identified, including NOD2 (Hugot et al., 2001; Ogura et al., 2001). However, the exact role of NOD2 and other NLR proteins in disease pathogenesis is still unclear. In their new paper, Oehlers et al. demonstrated the feasibility of using zebrafish as a model to study pathogen-induced IBD, and characterized components of the NOD1 and NOD2 signaling pathways that contribute to disease pathogenesis (Oehlers et al., 2011).
NLR protein family members are highly conserved in metazoans. In mammals, the NOD1 and NOD2 genes are highly expressed in the gut and hematopoetic cell lineages. Thus, Oehlers et al. first sought to identify zebrafish orthologs for NOD signaling components. Zebrafish orthologs for NOD1 and NOD2 had been previously identified (Laing et al., 2008). Expanding on these previous findings, Oehlers et al. used a bioinformatics approach to identify putative zebrafish a20, centaurin b1, erbin, grim-19, aamp and cd147 genes, which have all been identified as regulators of NOD signaling in mammals. Next, the authors evaluated the spatial and temporal expression patterns of nod1 and nod2 in zebrafish larvae. Using reverse-transcriptase PCR, whole-mount in situ hybridization and flow cytometric analysis, the authors demonstrated colocalized expression of nod1, nod2 and genes encoding components of the NOD signaling pathway throughout the gastrointestinal system of zebrafish larvae and in neutrophils. Together, these data demonstrate that the components of the NOD signaling cascade are present in zebrafish, and the expression patterns suggest that these elements might be relevant to gastrointestinal inflammation.
To establish whether these receptors are physiologically relevant, the authors complemented their expression studies with an in vivo pathogen-mediated model of IBD involving infection with the enteric pathogen Salmonella enterica. They knocked down nod1 and nod2 expression using gene-specific morpholinos, and then evaluated embryonic resistance to the infection. Knockdown of either nod1 or nod2 resulted in a significant reduction in bacterial clearance and survival, demonstrating an antibacterial role for these proteins. Although Nod2 is clearly involved in the immune response, further investigation suggested that Nod1 is more vital to zebrafish mucosal immunity. Following S. enterica infection, nod1 was shown to be highly expressed in neutrophils and was essential for neutrophil repopulation of the caudal hematopoietic tissue (CHT). These data are consistent with reports using mouse models of systemic bacterial infection, but conflicts with data that has been generated in mouse models of IBD. For example, a previous study involving an S. enterica Typhimurium-mediated mouse model of colitis showed normal disease progression in Nod1−/− and Nod2−/− mice (Geddes et al., 2010). However, consistent with the overall findings in the zebrafish model, a significant decrease in inflammation and an increase in bacterial burden was observed in Nod1−/−;Nod2−/−double-deficient mice (Geddes et al., 2010). Together, these findings suggest that NOD1 and NOD2 collaborate and/or have overlapping functions during gastrointestinal inflammation in mice, but that these two NLR proteins probably do not function through discrete mechanisms in zebrafish.
In addition to establishing physiological relevance for NOD signaling pathways in zebrafish, Oehlers et al. also investigated the mechanism of Nod1- and Nod2-mediated antimicrobial responses. In the intestinal tract of mammals, activation of NOD proteins instigates the production of reactive oxygen species (ROS) through increased expression of dual oxidase (encoded by DUOX2) (Lipinski et al., 2009). In zebrafish, duox expression in the intestinal epithelium has also been linked to ROS production and is associated with antibacterial activity (Flores et al., 2010). Here, Oehlers et al. observed reduced expression of duox in nod1 and nod2 morpholino-injected larvae compared with controls (in the absence of infection). This reduction was associated with increased intracellular bacterial burden in the nod1-knockdown animals when infected with S. enterica. However, no differences in bacterial burden were observed following knockdown of nod2, which is contrary to findings observed in in vitro mammalian studies. For example, in human intestinal epithelial cell lines, Listeria monocytogenes clearance was enhanced when both NOD2 and DUOX2 were overexpressed (Lipinski et al., 2009). Likewise, additional RNA-interference-based studies revealed that DUOX2 was required for NOD2 antibacterial activity (Lipinski et al., 2009). Together, these data suggest that NOD1 can facilitate DUOX2-mediated ROS production in zebrafish, whereas NOD2 is primarily responsible for this aspect of host defense in mammals.
The findings by Oehlers et al. demonstrate that NOD proteins and the signaling pathways that they initiate are important for antibacterial immunity in a zebrafish model that is relevant to IBD. As noted above, other members of the NLR protein family have been identified as either positive or negative regulators of the innate immune response in mice. In addition, several other NLR proteins have been shown to be relevant to intestinal inflammation in mouse models of IBD. Thus, the zebrafish models characterized in these studies have the potential to be developed into powerful tools to provide additional mechanistic insight into the in vivo contribution of NLR protein family members to IBD pathogenesis and host-pathogen interactions.