Gene expression analysis
In order to use CCO cells in bioassays, IL22Rα1 expression in these cells was confirmed using gene expression analysis (supporting material Figure S7). As a measure of mammalian IL-22 activity, upregulation in the expression of select response genes linked with antimicrobial activity and tissue repair function are typically monitored (Veas and Dubois, 2011, Wozniak, Hole, Yano, Fidel, & Wormley, 2014). Three response genes in cfIL-22 treated CCO cells were identified and assessed as a measure of cfIL-22 function: Fibronectin, for assessing tissue repair; interferon, a general immune response protein; and natural killer Lysin-1 an antimicrobial peptide involved in directly killing invading bacteria. These transcript levels of genes were assessed 24 hours after treatment with cfIL-22 or pBK plant background control at two concentrations (Figure 4). All response genes were assessed relative to a set of reference genes (alpha smooth muscle actin and alpha tubulin) established as suitable for this experiment. Interferon and NK-Lysin 1 showed significant increases in gene expression in both the 100 and 500ng/ml cfIL-22 treatments over pBK plant background control. Fibronectin was significantly increased in the 500ng/ml treatment.
DISCUSSION
This study serves as the first report supporting the use of plants to express bioactive therapeutic proteins with application for the aquaculture industry. Considering the inherent instability and rapid turnover of cytokines relative to most proteins, plants may offer a useful platform for expressing this class of protein. In addition, previous studies have reported expression of other fish IL-22 homologs with relatively low yields (Monte et al, 2011; Costa et al, 2013, Siupka et al, 2014, Qi et al, 2015). Using this well-described and tested transient expression system, plants are capable of expressing this protein at levels approaching 25.5 μg/gFW. Although the aim of this study did not focus on optimizing cfIL-22 expression levels, the transient plant expression platform is scalable and has been shown to support therapeutic protein production appropriate for commercialization (Sukenik et al, 2018; Holtz et al, 2015; D’Aoust et al, 2010). An alternative plant production platform in consideration of meeting a price acceptable to this commodity market are generating cfIL-22 stable transgenic plants wherein the recombinant therapeutic protein expressed in leaves or seeds could be directly incorporated into the fish’s diet without the need for extensive processing (Sissener et al, 2011).
While sequence analysis predicts ~20% identity between human and catfish IL-22, it is somewhat surprising how divergent the fish homologs are. Human shares 95.5% identity with resus macaque, while channel catfish shares only 83.1% identity with yellow catfish. The divergence is even more pronounced when comparing the second most related species. For human this is mouse with an 81% identity compared to channel catfish and zebrafish which share only 39.7% identity. Despite this sequence divergence among IL-22, all homologs do contain the IL-10 family signature, with ~80% identity in this region of the protein. Structural predictive software indicates the catfish protein also shares zebrafish and human IL-22’s globular structure (Siupka et al, 2014, Nagem et al, 2002; Supplement Figure 8).
The predicted monomer size of cfIL-22 based on amino acid sequence is 20kDa and is similar to that of other IL-22 homologs (Dudakov et al, 2015). Recombinant expression of this sequence in plants reveals two bands, ~20kDa and 24kDa (Fig. 2.2), which were confirmed by MS/MS sequencing to both be cfIL-22. Multiple bands have been previously reported for human IL-22 expressed in plants and associated with alternate glycoforms (Wilbers et al, 2016). While glycosylation was shown in this case as well as other recombinant fish IL-22 to not be necessary for its activity (Monte et al, 2011; Costa et al, 2013; Siupka et al, 2014; Qi et al, 2015), a Asn-Asn-Thr glycosylation site at aa 148 of cfIL-22 (Supporting material Figure S5) was confirmed to account for the two monomers observed (Supporting material Figure S6). A third band of ~44kDa that was consistently detected by western immunoblotting and confirmed by MS/MS analysis to be cfIL-22, is likely a dimer. This is consistent with human IL-22, which is known to produce dimers (Nagem et al, 2002; Neto et al, 2008).
After successfully producing catfish IL-22 in plants, it was important to establish that this recombinant protein had the associated predicted functional activity. To assess activity, cfIL-22 was purified to >90%. Although this three step purification scheme produced a low recovery (21%) of cfIL-22, achieving highly pure product is necessary for establishing accurate activity measurements and important for proof of concept of a plant host producing an active fish protein. As significant lipopolysaccharide levels can be associated withAgrobacteria -mediated production (gram negative soil bacteria) it was important to establish endotoxin levels in the purified protein products. Levels <0.1 EU/μg protein were typical and below levels accepted for commercialized recombinant proteins. Notably this endotoxin level aligns with commercial human IL-22 and lower than EU of mouse IL-22 product (Genscript, R&D Systems). Ensuring low or unmeasurable endotoxin was paramount in validating the bioactivity of our plant-made recombinant protein was indeed exclusive to catfish IL-22 (Gao and Tsan, 2003).
At the onset of this study, no in vitro bioassays to establish catfish IL-22 function had been reported. In general many cytokines are known to induce cell growth and division which is often used as an indicator of cytokine activity including IL-22 (Cai et al, 2010). The recombinant cfIL-22 notably increased the proliferation of catfish cells, highlighting the tissue preservation capabilities of this protein. Recombinant cfIL-22 dose dependent response induced proliferation over a broad concentration range with high levels of the cytokine resulting in expected cytotoxicity (Park et al, 2015; Park et al, 2011; Liang et al, 2010).
Activity for the majority of animal IL-22s including fish (Monte et al, 2011; Costa et al, 2013; Qi et al, 2015) has been validated using gene expression analyses. This along with the recent release of a channel catfish reference genome (Liu et al, 2016; Chen et al, 2016), made RTqPCR a credible approach for establishing the activity of our plant-made cfIL-22 protein. Using catfish fibroblast cells as our bioassessment model system, cfIL-22 upregulated expression of genes from three major categories important to combating disease. Consistent with the mammalian literature, cfIL-22 resulted in upregulation of genes encoding a tissue repair protein, an antimicrobial peptide, and a common innate immune protein. Fibronectin, an extracellular matrix protein involved in wound healing and tissue repair, expression was increased ~1-fold and corresponded with the well-known tissue preservation and repair activity of IL-22 (Sabat et al, 2014). Natural killer Lysin-1, involved in directly killing invading bacteria, is one of a number of antimicrobial peptides to be triggered by IL-22 and reported here to be increased ~2.5-fold. Finally, interferon, an important protein involved in modulating host immunity to different pathogens, was markedly upregulated (~2-fold) by cfIL-22. These findings are consistent with other studies of fish IL-22 in rainbow trout (Monte et al, 2011), turbot (Costa et al, 2013), zebrafish (Siupka et al, 2014) and mullet (Qi et al, 2015).
Channel catfish (Ictalurus punctatus ) is a warm water species and is currently the number one farmed fin fish in the United States with Alabama, Arkansas, and Mississippi accounting for ~60% of total US produced aquaculture products (Saroglia and Liu, 2012). Stress and disease-induced losses are estimated to account for more than $70 million annually (Welker, Lim, Yildirim-Aksoy, Shelby, & Klesius, 2007; USDA, 2011). There is opportunity for developing novel tools to aid catfish farmers in managing fish health. A notable advantage of producing a recombinant therapeutic protein for fish in plants is the ability to incorporate the active therapeutant directly into a component of their diet (e.g. soybean, corn) that is readily consistent with medicated feed applications currently used in the aquaculture industry.
Another significant advantage to exploring protein-based therapeutics in general is they offer greater selectivity and specificity over currently used chemicals and antibiotics. A cfIL-22 therapeutant should not be active on other animals or organisms comprising the aquaculture environment providing better environmental safety profiles. In fact cfIL-22 was tested in a cell line of a related species to channel catfish (walking catfish, G1B) and cfIL-22 was not active. Furthermore, with economic incentives to increase fish density that further complicates disease management and increases antibiotic usage in the aquaculture industry, recombinant immune proteins could offer a safer alternative. With increased consumer demand for antibiotic-free food, and interest in avoiding antibiotic resistant bacterial strains associated with food production, alternatives to antibiotics for the aquaculture industry are much needed. Interestingly, one of the most valuable traits of IL-22 as a therapeutic target is potentially its associated antimicrobial activity.
While more fully defining the antimicrobial effects of cfIL-22 is required, this study is the first to suggest that at least one antimicrobial gene (NK1 lysin) is induced by the catfish homolog of IL-22. Unfortunately with limited in vitro based tools to study catfish immunity, the commercially available channel catfish fibroblast cell line used in this study is not ideal. There are IL-22 receptors located on fibroblasts in mouse cells however it is known that the majority of IL-22 receptors are located on epithelial cells (Sabat et al, 2014; McGee et al, 2013). Therefore, to adequately evaluate the function of cfIL-22 in channel catfish our group is in the process of developing epithelial cell lines.
In this study, we successfully produced cfIL-22 in plants at adequate amounts and purity to enable biochemical characterization and to conductin vitro bioactivity of this proposed therapeutant. Biochemical analysis confirmed signal peptide processing and dimer formation. Bioassays designed to test cfIL-22 function confirmed both the tissue preservation and repair properties of IL-22 as well as immune stimulation and antimicrobial production. These findings support plant made recombinant catfish interleukin-22 as a possible therapeutic for the aquaculture industry and support further analysis of this protein for therapeutic use.
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TABLES
FIGURE LEGENDS