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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FIGURE LEGENDS