NOD2 Mediated Host-Commensal
Interactions in Stress-induced
Rainbow Trout (O. mykiss)
Josh Trujeque, Luca Tacchi, Rami Musharrafieh, Erin Larragoite, Irene Salinas
Center for Evolutionary and Theoretical Immunology, Department of Biology,
University of New Mexico
1. Introduction
When searching for principles of war, Vegetius stated that if you wish for peace you must
prepare for war. This is evident on the battlefield within all organisms, and an organism’s
defense and offense is known as the immune system. The immune system is a vast network of
cells, tissues, and organs that protects organisms from foreign invaders that may result in
infection and disease. The immune system must distinguish between foreign invaders to launch
attacks against and host tissue to ignore. The capability to create a state of sufficient biological
defense against infection and disease is known as immunity. Immunity is established by the
immune system, where there are two major divisions in vertebrates, referred to as the adaptive
immune system and the innate immune system [Janeway et al. 2001]. The adaptive immune
system is characterized by antigen specificity, identification of self-vs. nonself, and the
establishment of immunological memory by highly specialized systemic cells such as B and T
cells [Janeway et al. 2001]. The adaptive immune system has the job of allowing certain
organisms to exist with the host. In contrast, the innate immune system is based on the
recognition of general patterns that are conserved in microbial or pathogen associated epitopes
[Janeway et al. 2001]. These nonspecific patterns are known as pathogen associated molecular
patterns (PAMPs) and are recognized by pattern recognition receptors (PRRs) [Chang et al.
2011]. PRRs can be found on cell membranes extracellularly as well as intracellularly in
endosomes [Janeway et al. 2001].
The front lines for the immune system are the points of contact of every host within the
environment. These points of contact are the main portals of entry for pathogens. Different host
differ in their environment and the nature of surfaces that interact with the environment, resulting
in extremely diverse and complex interactions. The majority of vertebrates acquired mucosal
epithelia to cover most of their external surfaces. Mucosal epithelia are therefore the first point of
contact and consist of several barriers of defense. Mucosal surfaces serve, not only as a physical
barrier, but as a biochemical barrier and a functionally interactive barrier [Sansonetti 2006]. This
functionally interactive barrier is referred to as the mucosal-associated lymphoid system
(MALT), which is active in its protection against pathogens while actively suppressing its
defense mechanisms against non-pathogens [Maynard et al. 2012, Kamada et al. 2013, Cerutti et
al. 2013].
MALT is characterized by the epithelium on which multiple mucosal layers are present
[Maynard et al. 2012, Kamada et al. 2013, Cerutti et al. 2013]. The epithelium consists of a layer
or layers of epithelial cells that form a continuous physical barrier, held together by
transmembrane proteins such as tight junction and zona adherens proteins, and it is constantly
regenerated [Sansonetti 2006]. Although epithelial cells are held closely by tight junctions and
adherens, disruptions of these proteins can cause increased permeability of this barrier, making
an organism more susceptible to invasion by opportunistic bacteria [Maynard et al. 2012, Hooper
et al. 2010, Sansonetti 2006]. An additional component of the epithelium is specialized epithelial
cells called goblet cells that secrete heavily glycosylated proteins referred to as mucins [Maynard
et al. 2012, Hooper et al. 2010, Janeway et al. 2001]. Mucins solubilize in water to form mucus,
and because they are heavily glycosylated, they can act as decoys for bacterial adhesion
preventing binding of bacteria to the epithelium which is characteristic of mucosal surfaces
[Linden 2008]. In mammals, the inner layer of mucus is comprised of sub layers that exemplify
how mucus acts as a barrier to bacteria. The inner layer near the apical surface consists of much
less bacteria, compared to the outer layer, thus showing how the inner layer can resist bacteria
[Hooper et al. 2010]. Epithelial cells are not merely the bricks that form epithelial barriers but are
active players in mucosal immunity. In addition, epithelial cells can be stimulated by signaling
molecules called chemokines and cytokines, such as IL-17A, IL-1, and IL-6, which activate
production of antimicrobial peptides [Ramirez-Carrozzi et al. 2011]. Antimicrobial peptides are
expressed constitutively and are mostly hydrophobic and basic, making them a broad-spectrum
peptide because of the capability to disrupt the cell wall of a broad range of bacteria [Hooper et
al. 2010, 26]. When an organism is lacking in the production of mucus and/or antimicrobial
peptides, the organism cannot maintain the barrier against bacteria and therefore has an increased
susceptibility to bacterial invasion and ultimately inflammation [Hooper et al. 2010]. This entire
complex of epithelium and mucus is referred to as the mucosa and acts as a microbial buffer, like
the gastrointestinal tract in humans, and other mucosal surfaces [Maynard 2012, Kamada et al.
2013].
Moreover, certain organisms have co-evolved with particular vertebrates, leaving the host
unaffected, within a hospitable nutrient rich environment provided by the host which is known as
commensalism. In mammals, intestinal bacteria in the mucosa can exist to be mutualistic and
break down certain polysaccharides, and synthesize beneficial natural products such as vitamins
that the host is unable to do [Maynard et al. 2012, Hooper et al. 2010]. Commensals are certain
types of intestinal bacteria that have co-evolved with vertebrates to form a relationship with the
host, yielding reciprocal host-bacterial regulatory mechanisms, excluding the growth of as well
as stimulating immune responses to pathogenic bacteria [Hooper et al. 2010, 25]. In return,
commensals receive a stable nutrient-rich environment [Hooper et al. 2010]. For example, there
are over 100 trillion organisms that reside in the gastrointestinal tract of mammals, mostly
bacteria in the distal ileum and colon that [Maynard et al. 2012]. It is estimated that there are
over 1,000 species of intestinal bacteria, with only a small portion existing in any one individual,
exemplifying how diverse the intestinal bacterial composition can be in any given individual
[Maynard et al. 2012]. The immune system of vertebrates needs to discern between pathogens
and commensals to maintain homeostasis [Janeway et al. 2001]. Commensals, in turn, send
signals to the host necessary for epithelial turnover, the development of innate and adaptive
immune cells [Maynard et al. 2012, Hooper et al. 2010].
In order to benefit from mutualistic relationship with microorganisms. The immune
system faces important challenges. It needs to be effective at defending the host against
pathogens while being hypo-responsive against commensals [Sansonetti 2006a, Sansonetti
2006b]. The immune system needs to be hyporesponsive against commensals in order to perturb
unnecessary inflammation from an overactive immune response and to ensure that the metabolic
networks beneficial to the host are not altered [Abren 2010, Sansonetti 2006a, Sansonetti 2006b].
Whereas the adaptive immune system’s job is to learn self, these commensal bacteria can be
viewed as ‘extended self’, allowing it to be hyporesponsive against commensals so they can
colonize [Maynard et al. 2012]. When the adaptive immune system is unable to delineate
between pathogens and commensals that may share similar antigens, an immune response will be
generated, causing inflammation that has a potential to be chronic [Nussbaum and Locksley
2012]. The ‘frustrated commensal’ model describes that the adaptive immune system's confusion
between pathogens and commensals perpetuates inflammation [Nussbaum and Locksley
2012]. Furthermore, host commensal interactions are maintained by cytokines and chemokines,
which are chemicals that relay particular messages between cells in order to initiate or terminate
an immune function [Sansonetti 2006]. The host uses chemokines and cytokines to induce a
tolerogenic state among the immune related cells that would normally cause inflammation
[Sansonetti 2006]. This tolerogenic state happens via microbe-associated molecular patterns
(MAMPs) and their critical interaction with PRRs in maintaining mutualistic or symbiotic
relationships between the host and symbiont [McFall-Ngai et al. 2011]. Recognition of MAMPs
by PRRs activate a special subset of T cells known as regulatory T cells (Tregs) that suppress the
responses of other immune cells that would try to attack the resident bacteria and induce
inflammation [Janeway et al. 2001]. In addition to Tregs, another mechanism that is associated
with sustaining a tolerogenic state is transforming growth factor beta (TGF-β). TGF-β is a
multifunctional cytokine that is a part of many cellular pathways, including cell growth,
apoptosis, fibrosis, differentiation, and immune responses [Rabinowitz et al. 2013]. Although
TGF-β is known to activate Tregs and inhibit effector T cell proliferation, it is known to have
proinflammatory activity by the activation of Th17 cells which are another subset of T cells
[Rabinowitz et al. 2013]. In contrast to commensals that create a tolerogenic state using
cytokines and chemokines, bacteria that cannot return the dialogue are eliminated by
inflammatory responses [Nussbaum et al. 2012]. Thus, certain bacteria have evolved to move
into compartments of the host to evade host immune responses, therefore becoming a virulent
agent [Nussbaum et al. 2012]. There are two outcomes of non-commensal bacteria, they are
eliminated or they move into host tissue [Nussbaum et al. 2012]. Sometimes, bacteria invade
host cells and have the ability to live in the cytosol of these cells.
Many factors can alter the composition of intestinal bacteria such as diet, stress, and
antibiotics, causing a malfunction in the regulatory pathways of the mucosal immune system,
also known as dysbiosis [Maynard et al. 2012, Cerutti et al. 2013, Honda et al. 2012] . In some
cases, this is caused by the breakdown of immune barriers, resulting in bacterial translocation
[Maynard et al. 2012, Kamada et al. 2013, Cerutti et al. 2013]. For example, E. coli and C.
difficile have been shown to increase the permeability of the epithelial barrier by disrupting the
tight junctions of those cells [Sansonetti 2006]. Although most bacteria are mutualistic there are
some species that were once beneficial at a certain point in time but can opportunistically
become a pathogen. For example, Enterococcus faecalis and Bacteroides fragilis are prominent
members of the human intestine which can opportunistically invade, especially in
immunodeficient individuals [Hooper et al. 2010]. In mammals, bacterial translocation of
commensal bacteria has been shown to be crucial in the initiation of chronic stress-induced
colonic inflammation, and CD [Maeda 2005, Inohara 2005]. The mucosa serves to promote and
maintain the relationship between commensals but must also control them because they can still
translocate and cause infection or inflammation under unbalanced conditions [Maynard et al.
2012, Hooper et al. 2010, Honda et al. 2012]. In contrast, some bacteria have the ability to
disrupt the mucus layer such as Helicobacter pylori. This bacterium, increases the mucus pH
causing a decrease in the viscosity of the mucus, allowing it to penetrate the mucus layer
[Hooper et al. 2010].
There are several factors that are associated with maintaining a functional mucosal barrier
such as diet, hygiene, and stress [Maslanik et al. 2012]. When stress is induced in mammals,
sympathetic innervations cause mast cell degranulations that can alter the composition of
commensal bacteria and can lead to a compromised intestinal barrier [Maslanik et al. 2012]. This
change in commensal bacterial composition can alter immune functions [Maslanik et al. 2012].
These commensal bacteria are thought to be translocating in mucus producing goblet cells, using
the structure of the goblet cell to be engulfed [Maynard et al. 2012, Kamada et al. 2013].
Dysbiotic flora leads to inflammation of the intestine, and depending on the pathogen and
location, can result in inflammatory bowel diseases (IBD) [Maynard et al. 2012]. It has been
discovered that patients with IBD have higher numbers of intestinal bacteria associated with the
epithelial cell surface, indicating a malfunction in the mechanisms that control that interaction
[Hooper et al. 2010]. There are many inflammatory bowel diseases such as Crohn’s disease (CD)
which are not well understood, but it is known that CD has environmental and genetic factors
that lead to an uncontrolled immune response against commensal bacteria [Perez et al. 2010].
Furthermore, certain mutations have been associated with inflammatory bowel diseases, like CD
which have been shown to be linked to a dysfunctional nucleotide-binding oligomerization
domain 2 (NOD2) gene [Hooper et al. 2010]. Also, TGF-β and its receptor are overexpressed in
the intestines of CD patients [Rabinowitz et al. 2013]. CD is not caused by a particular pathogen
but rather a multitude of different pathogenic bacteria that cause a similar overlapping phenotype
known as CD [Campbell et al. 2012]. These overlapping phenotypes associated with CD
exemplify interactions with a dysbiotic flora.
Within the innate immune system, intracellular PRRs are the main molecules involved in
interacting with MAMPs and in mammals are three main families: Interferon (IFN)-inducible
proteins, retinoic acid-inducible gene 1 (RIG-1)-like receptors (RLRs), and nucleotide-binding
oligomerization domain (NOD)-like receptors (NLRs) [Chang et al. 2011]. These PRRs are used
to determine the ‘pathogenicity’ of bacterial by sensing their PAMPs [28]. Specifically, NLRs
are expressed most in the cytosol of epithelial cells and immune cells such as macrophages,
dendritic cells, and neutrophils, but are expressed in most tissues [Maynard 2012, Chang et al.
2010]. NLRs have been associated with autoimmune diseases and also sensing of bacteria and
viruses [Fritz et al. 2006]. NLRs consist of three domains: at the N-terminus is an effector
binding domain (EBD), in the center is the NOD also known as NACHT domain, and at the Cterminus is a leucine-rich repeat (LRR) domain [Chang et al. 2010, Fritz et al. 2006, Laing et al.
2008]. The EBD is involved in protein to protein interactions but vary in type between NLR
subgroups which can have a caspase recruitment domain (CARD) or pyrin domain [Chang et al.
2011, Fritz et al. 2006, Laing et al. 2008]. The NACHT domain which stands for domain present
in Naip, CIITA, HET-E (plant het product involved in vegetative incompatibility) and TP-1
(telomerase-associated protein 1) exhibits NTPase activity and is closely related to the
oligomerization module that is needed to activate effector molecules downstream [Janeway et al.
2001, Chang et al. 2011, Fritz et al. 2006 ]. LRRs are also involved in protein to protein
interactions as well as recognition of PAMPs [Janeway et al. 2001].
The NLR family encompasses two major receptor subfamilies referred to as NALP and
NOD. Although NALPs have not been defined better, NODs have been shown to be associated
with intestinal epithelial cells and macrophages within the mammalian gut [Laing et al. 2008].
Within these cells, NOD1 and NOD2 are both highly expressed and with oligomerization of
these receptors that are mediated by their NOD domains [Laing et al. 2008, Hou et al. 2012b].
NOD2 has been focused on more because NOD2 mutations have been linked to enhanced
susceptibility to CD and mice deficient in NOD2 are associated with aggressive Th1 responses
that promote tissue damage and inflammatory diseases [Denise et al. 2005, Watanabe et al.
2004]. NOD2 is localized in the cytosol of cells and recognizes muramyl dipeptide (MDP) which
is a component indicative of Gram-positive bacteria [Laing et al. 2008]. In mammals, NOD2 is
capable of triggering multiple effector signaling pathways in the innate and adaptive immune
system that resist microbial and viral pathogens [Chang et al. 2011, Kobayashi et al. 2005].
NOD2 is characterized by two CARD domains on the N-terminus, a central NOD or NACHT
domain, and a LRR domain on the C-terminal of this receptor [Chang et al. 2011, Laing et al.
2008]. The two CARD domains interact with each other and are responsible for the activation of
the NF-kB, which is central to a critical pathway involved in proinflammatory responses that
ultimately results in production of chemokines, cytokines, and antimicrobial peptides [Maynard
2012, Chang et al. 2011]. When NF-kB is activated, it is formed into dimers bound by IkB
complexes which holds the dimer and cannot be released until it is phosphorylated after
ubiquitylation and proteasomal degradation downstream of PRRs [Maynard et al. 2012]. When
commensals come in contact with intestinal epithelial cells, they inhibit the phosphorylation and
ultimate degradation of IkB, therefore inhibiting the continuation of the NF-kB pathway and is
an example of host-commensal interactions that suppress the immune system and inflammation
[Maynard et al. 2012].
The mammalian NLR family has been found in lower vertebrates and invertebrates. The
NOD subfamily of NLRs has been highly conserved throughout evolution, being established
before the divergence of teleost fish from the tetrapod lineage [Laing et al. 2008]. Specifically,
human NODs 1-5 are orthologs to many ectothermic teleost fish such as zebrafish (Danio rerio),
grass carp (Ctenopharyngodon idella), orange grouper (Epinephelus coioides), channel catfish
(Ictalurus punctatus), japanese flounder (Paralichthys olivaceus), rohu (Labeo rohita), and
rainbow trout (Oncorhynchus mykiss) [Chang et al. 2011, Fritz et al. 2006, Hou et al. 2012, 30].
Sequences for the nine different teleost species are available in GenBank through NCBI. Teleost
fish NODs and NALPs are referred to as NLR-A, and NLR-B respectively [Laing et al. 2008]. In
goldfish, the highest mRNA levels of NLR-A2 (NOD2) occurred in neutrophils and spleen cells
as compared to zebrafish which had the highest mRNA levels of NLR-A2, indicating a strong
similarity to the expression of NOD2 in human intestinal cells [Chang et al. 2011, Laing et al.
2008, Hou et al. 2012, Xie et al. 2013]. Although NOD2 is detectable in gill, thymus, brain, skin,
muscle, liver, spleen, head kidney, intestine, and heart, the RTG-2 cell line of rainbow trout had
the highest level of expression in the muscle and liver of trout NOD2 (trNOD2) [Chang et al.
2011].
Teleost fish skin has special characteristics and is in relation with particular pathogens
and commensals. The skin of teleost fish is known to be a part of the mucosal-associated
lymphoid tissue (MALT), often referred to in mammals [Salinas 2011]. In teleost fish, the
immune related tissues within mucosal epithelium of teleost fish are known as skin-associated
lymphoid tissue (SALT), that was first described in mammals [Streilein 1983]. In contrast to
mammals, the teleost SALT can still divide and is nonkeratinized [Salinas 2011]. Within teleost
SALT exists immune related cells such as macrophages, granulocytes, mast cells, dendritic cells
and plasma cells [Davidson et al. 1993a, Herbomel et al. 2001, Iger et al. 1988]. Additionally,
Staphylococcus aureus has been found to survive within its hosts cells, known as a facultative
intracellular pathogen [Fraunholz et al. 2012]. Staphylococcus aureus has been reported to
persist inside phagocytes or endothelial cells for prolonged periods, possibly indicating an
evasive strategy by avoiding detection by professional phagocytes [Fraunholz et al. 2012]. S.
aureus causes skin diseases in humans thanks to its ability to be internalized by human
keratinocytes [Kintarak 2004]. Staphylococcus warneri has been isolated in rainbow trout and is
able to colonize skin epithelial cells and exist as a intracellular commensal bacteria [Gil et al.
2008]. An example of a pathogenic bacteria related to aquatic vertebrates is Vibrio anguillarum
[Frans et al. 2011]. This bacteria can infect teleost fish, causing a fatal haemorrhagic septicaemic
disease, called vibriosis [Frans et al. 2011]. The role of NOD2 in relation to commensals like
Staphylococcus warneri and pathogens like Vibrio anguillarum have yet to be determined.
Furthermore, teleost fish like rainbow trout use alternative splicing of NODs in
inflammatory events, with some alternative transcripts proposed to be competitive inhibitors of
normal NOD2 [Chang et al. 2010, Laing et al. 2008]. When NOD2 is activated in teleost fish,
RIP-like-interacting CLARP kinases (RICKs) are recruited, which then interact with CARDCARD domains [Hou et al. 2012]. This interaction leads to the activation of NF-kB that
ultimately increases expression of IL-1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α), and IFN-γ
[Hou et al. 2012, Hou et al. 2012]. In human NOD2, the TLR domain binds MDP and because
teleost fish have highly conserved LRR regions as compared to humans, NLR-A is thought to
retain this function [Laing et al. 2008]. In terms of functional assays we know that in teleost fish,
NOD2 expression is enhanced by PolyI:C, IL-1β, and IFN-γ, lipopolysaccharides, and
peptidoglycan [Hou et al. 2012, 30]. Furthermore, goldfish macrophage cultures that were
exposed to two different heat-killed pathogens, Aeromonas salmonicida and Mycobacterium
marinum, that showed increased levels of expression of goldfish NOD1 (gfNOD1) and gfNOD2
compared to control cultures. To date, the relationships between NOD2 and commensals in
teleost mucosal surfaces has not been investigated [Xie et al. 2013].
Thus far, we have explored mucosal surfaces of vertebrates as points of contacts to
microbes and potential pathogens, understanding that within mucosal immunity, the innate
immune system is critical in sustaining a tolerogenic and symbiotic state against commensal and
mutualistic bacteria. This tolerogenic and symbiotic state relies upon the dialogue of chemokines
and cytokines that are upregulated by signal cascades determined by MAMP-PRR interactions
upon the mucosal epithelium. As compared to a symbiotic state, commensal bacteria in
vertebrates can compromise the host by a shifting bacterial composition that can increase the
number of bacteria that can potentially translocate into the host. When the particular balance of
commensal or mutualistic bacteria between opportunistic or potentially pathogenic bacteria is
disturbed, the host is in dysbiosis. Dysbiosis in vertebrates can lead to the breakdown of physical
and immune barriers, allowing bacteria to translocate inside of the host. When bacteria
translocate, immune responses are generated, causing inflammation and ultimately inflammatory
diseases. An example of an inflammatory disease is CD, which is a term that encompasses many
types of inflammation, caused by factors associated with dysbiosis. It had been discovered
through genome sequencing of CD patients, that there was a shared nonfunctional NOD2 gene,
associating with an observation that these patients have a higher bacterial load on the apical
surface of intestinal epithelial cells. From this correlation, it was found that the NOD2 gene is an
innate intracellular PRR that recognizes specific bacterial patterns, indicating how important the
innate immune system is in maintaining a dialogue between the host and the microbial
community in the environment to sustain a tolerogenic and symbiotic state. The NOD2 gene is
characterized within a family of innate PRRs known as the NLR family, with NODs and NALPs
being subfamilies. With the NLR family being highly conserved amongst many vertebrates such
as teleost fish, it is possible to further understand the role of PRRs, like NOD2, as a member of
the innate immune system in the context of a functionally interactive barrier that is creating or
suppressing an immune response. Specifically, orthologs of the NOD genes of humans have been
found through sequencing of many teleost fish such as rainbow trout and zebrafish, allowing new
insights about the various functions of the NOD subfamily in relation to mucosal immunity. In
fact, zebrafish gut NOD2 has been proposed as a model for the study of CD [Oehlers et al.
2011]. Currently, the location and role of NOD2 in the skin of teleost in regards to commensal
and stress-induced bacterial translocation is unknown. The goal of my research project is to first
determine if NOD2 is expressed in the skin of rainbow trout, which cells are the ones expressing
it, its role in sensing intracellular commensal bacteria in the skin and finally, how stress affects
NOD2 expression in trout skin.
2. Materials and methods
2.1 Fish in vivo stress model
Rainbow trout (Oncorhynchus mykiss) will be obtained from the Libosa Springs
Hatchery in Pecos, New Mexico. Fish (mean weight 200g) will be starved 48 hours prior to the
start of the experiment. The skin samples (n=6) (1cm2) will be taken from the left side of the fish,
below the dorsal fin and right above the lateral line in order to maintain consistency. The mucus
layer will not be disturbed. Each piece of skin will be frozen in OCT and snap frozen in liquid
nitrogen. For the stress experiment, three experimental groups will be used: 1) control trout (pretransport), post-transport with salt (2.5g/L) (PTS), and post-transport without salt (PTNS). The
stress transport experiment consists of a 5 hours drive in a truck for both PTS and PTNS.
2.2 Skin Cryosections and Fluorescent In situ Hybridization (FISH)
Tissue samples from the trout skin will be obtained and sectioned in a cryostat, which is a
device used to cut histological slides up to five microns thin while maintaining low cryogenic
temperatures of -20C to sustain the integrity of the tissue samples. The microtome cryostat will
be used to cut 5m-thick sections from each cryoblock and stored at -80C until further use.
Fluorescence in situ hybridization (FISH) will be used to identify which cells express
NOD2 in the skin of rainbow trout. This method is comprised of oligonucleotide probes each
with a fluorescent label that bind a target transcript of interest, in this case NOD2, to produce a
fluorescent signal. The FISH method allows visualization of RNA molecules at the subcellular
level which can be analyzed and captured via fluorescence microscopy. FISH probes must be
designed to bind to a specific sequence of mRNA that is unique to that gene. For the NOD2
probe, we will use NCBI’s basic local alignment search tool (BLAST) in order to find all teleost
and other vertebrate NOD sequences. All NOD2 sequences will be aligned using multiple
sequence alignment by CLUSTALW. This allows for identification of regions of local similarity
between sequences, calculating the statistical significance of similarities between numerous
species. Since trout NOD2 has two splice variants, NOD2a and NOD2b, we will target the
mRNA of NOD2a because it is the predominant splice variant. The sequence of the NOD2a was
obtained from the GenBank (accession No, HM113906) and the probe was labeled with Cy3 in
the 5’ end of the oligonucleotide [Chang et al. 2010]. The BLAST allowed us to determine a
particular sequence that is located on the second CARD domain of the NOD2a splice variant of
rainbow trout. As a negative control, a Cy3-labeled antisense probe will be used instead of the
NOD2 probe. Cryosections will be air dried, fixed in 10% formalin, washed twice with PBS, and
permeabilized overnight in 70% ethanol. Probes were then hybridized in SSC and formamide
onto the slides and incubated in the dark overnight. Slides were each washed twice with SSC and
PBS then hybridized with DAPI, washed once with PBS, and mounted with slide covers and
mounting media.
After hybridization, samples will be observed under a Nikon Ti fluorescent microscope.
Images will be analyzed using Elements Advanced Research Software in conjunction with the
Nikon Ti Fluorescent microscope.
2.3 Bacterial strains
The bacterium, Staphylococcus warneri, was isolated from the skin of control adult
triploid rainbow trout. The skin mucus was first removed with a sterile cell scraper. After
spraying the skin with ethanol, a 2 cm2 section of skin was dissected and placed in HBSS
containing penicillin and streptomycin. After incubation and shaking at room temperature for 2h,
the skin sample was transferred to a Petri dish containing HBSS without antibiotics. At this point
the skin was finely minced (over 100 times). The cells were further lysed using a 1ml syringe.
The suspension was vortexed for 1 min and then centrifuged at 3000g, 10 min. The pellet was
resuspended in sterile phosphate buffered saline and 10ul of the suspension were plated in either
LB or TSA plates. Bacteria were allowed to grow for 3 days. Three different types of colonies
could be observed. Out of these, one of them was sub-cultured into TSA plates. The pure
cultures were identified at the Tricore laboratories (Albuquerque, New Mexico) by means of
Gram stains and MALDI-TOF. These tests revealed that the isolate is the Gram positive cocci,
Staphylococcus warneri. The identity was further confirmed by PCR using specific 16s rDNA
primers, cloning and sequencing.
The bacterium, Vibrio anguillarum, is a known Gram negative teleost pathogen. This
bacterium was kindly donated by Dr. Debra Milton at the Southern Research Institute.
2.4 In vitro skin explant cultures
Control trout (n=5) will be used for the in vitro experiments. A piece of skin (0.5cm2)
will be obtained from the same site as explained above. The skin explants will be surface
sterilized for 30 s twice in 70% ethanol followed by two washes in PBS under sterile conditions.
24-well plates will be used for the in vitro stimulation of the skin explants. Five experimental
groups were used: control (unstimulated), exposed to 106 cfu of Vibrio anguillarum, exposed to
102 cfu of Staphylococcus warneri, exposed to 104 cfu of S. warneri. Skin explants were
exposed to these treatments for 4, 24 or 48h. At this points, skin tissue was collected and placed
in Trizol. RNA will be purified and then the RNA can be further separated so that the mRNA can
be utilized. The mRNA is used because that is the RNA that indicates what is being transcribed
into proteins. Next, a reverse transcriptase (RT) enzyme is used to transcribe the coding DNA
(cDNA) strand which can then be used for qPCR. After the qPCR, the data will be analyzed in
order to determine the fold change relative to the elongation factor alpha (EF-⍺) gene that is
always expressed in all cells.
2.5 Expression of NOD2a, NOD2b, and NF-kB in the in vitro skin explant and in vivo stress
model
To determine the expression of NOD2a, NOD2b and NF-kB in the skin of rainbow trout
when exposed to Staphylococcus warneri and Vibrio anguillarum we will use real time
quantitative polymerase chain reactions (RT-qPCR). RT-qPCR was used to determine the
abundance of NOD2a, NOD2b, and NF-kB in O. mykiss skin tissues. The expression of NOD2a
and NOD2b in the skin of PTS and PTNS rainbow trout was also quantified using RT-qPCR.
The RT-qPCRs were performed in triplicate and each contained 3 μl of a diluted cDNA template,
12.5 μl of Power SYBR Green PCR master mix (2× Applied Biosystems), and 100 nM forward
and reverse primers in a 25 μl reaction volume. The amplification profile consisted of an initial
denaturation step at 95°C for 10 min, and then 30 cycles of 95°C for 15 s and 60°C for 1 min
followed by melting (dissociation stage) from 72°C to 95°C in an ABI Prism 7000 (Applied
Biosystems) sequence detection system. A negative control (no template) reaction was also
performed for each primer pair. A sample from the serial dilution was run on a 2% agarose gel
and stained with RedGel Nucleic Acid Stain (Biotium) and viewed under UV light to confirm a
band of the correct size was amplified. A melting curve for each PCR was determined by reading
fluorescence at every degree between 72ºC and 95ºC to ensure only a single product had been
amplified. Rainbow trout elongation factor EF-1α was used as control gene for normalization of
expression.
The relative expression level of the genes was determined using the Pfaffl method (Pfaffl
2001). Efficiency of the amplification was determined for each primer pair using serial 10 fold
dilutions of pooled cDNA, performed on the same plate as the experimental samples. The
efficiency was calculated as E = 10 (-1/s) where s is the slope generated from the serial dilutions,
when Log dilution is plotted against CT (threshold cycle number). Primers were designed to
have a Tm of 55°C, and where possible, to cross an exon-exon junction to avoid amplification of
genomic DNA. Exon-intron junction sites were determined by comparing the rainbow trout
cDNA with genomic sequence for orthologous genes from other vertebrates obtained from
NCBI’s basic local alignment search tool (BLAST) (http://blast.ncbi.nlm.nih.gov/).
3. Results
3.1 Localization of NOD2 using FISH
Analysis of rainbow trout skin using FISH (Figure 1) shows the expression of NOD2a in
epithelial cells and goblet cells present near the apical surface of the trout. In the PTS trout, there
was a higher localization signature, indicating increased expression in relation to the control. In
the PTNS trout, expression was higher than the control trout but lower than the PTS trout.
Fig 1. Diagram of FISH in the skin of rainbow trout, indicating location of NOD2 in epithelial cells (EC)
and goblet cells (GC). A: control B: PTS C: PTNS
3.2 Expression of NOD2a, NOD2b, and NF-kB in the in vitro skin explant and in vivo stress
model
Analysis of the in vivo stress model of rainbow trout using RT-qPCR showed that
trNOD2a expression had a decreased fold change of 1.5 in PTNS trout, and an increased fold
change of 1.5 in PTS trout. trNOD2b expression was not significant in trNOD2b, but a
significant fold change increase of 2 in PTS trout (Figure 2).
Fig 2. Expression of two trout NOD2 splice variants
using RT-qPCR within an in vivo stress model in the
skin of trout. Relative expression was normalized
against expression of EF-1α. Asterisks indicate
where expression is significant (P < 0.05).
Furthermore, expression of the in vitro skin explants of rainbow trout via RT-qPCR showed a
dose dependent relationship that was consistent between trNOD2a, trNOD2b, and NF-kB (Figure
3). Expression of trNOD2a at the lowest dose had a 3 fold decrease during the first 4h, a 2 fold
increase at 24h, and a 2 fold decrease at 48h. Expression of trNOD2b at the lowest dose had a
twofold decrease during 4h, a twofold increase at 24h, and a 3 fold decrease at 48 hours. The
expression of trNOD2a and trNOD2b was not significantly affected by the V. anguillarum,
consistent as a positive control because NOD2 is only known to sense Gram positive bacteria,
not Gram negative bacteria such as V. anguillarum. Expression of NF-kB at the lowest does had
a tenfold decrease at 4h, a 15 fold increase at 24h, and a fivefold decrease at 48h. NF-kB had a
much higher magnitude of fold change as compared to both trNOD2 splice variants but followed
the same trend.
4. Discussion
In this study, we examine NOD2, an innate intracellular pattern recognition receptor that
activates NF-kB in rainbow trout, O. mykiss [Chang et al. 2010].
We first looked at the
expression of two trout NOD2 splice variants in an in vivo stress model, where NOD2 was
downregulated in PTNS trout and upregulated in PTS trout, possibly indicating active
mechanism of downregulation by the dominant microbes in PTNS trout.
In addition, we examined the expression of two trout NOD2 splice variants and NF-kB in
trout skin explants in vitro. These skin explants were exposed to either S. warneri or V.
anguillarum at various doses. Human NOD2 is known to recognize gram positive bacteria, and
because trout NOD2 is a homolog, we would expect trout NOD2 to only recognize S. warneri
and not V. anguillarum [Chang et al. 2010]. This was confirmed through analysis of RT-qPCR
where NOD2 and NF-kB expression was significantly affected by S. warneri and not
significantly affected by V. anguillarum. Moreover, the initial downregulation of NOD2 and NFkB by S. warneri during 4h indicates an active innate immune suppression, characteristic of a
commensal bacteria. But during the 24h time period, the increased fold change of NOD2 and
NF-kB indicates a recognition of this bacterium as a pathogen. These results further support the
‘frustrated commensal’ theory, but the mechanism of action is unknown [Nussbaum et al. 2012].
Furthermore, we examined the location of trout NOD2a, due to the predominant
expression of this spice variant, in the skin of rainbow trout using FISH. Although expression
was conclusive in epithelial and goblet cells, there was some expression near the muscle but
conclusive images have yet to be captured. It has also has been observed that S. warneri grows
better under PTS conditions. This observation may support how NOD2 is upregulated in PTS
trout, because it is sensing more of this gram positive bacteria. This is in opposition to PTNS
trout where we observed a decreased fold change, indicating less gram positive bacteria resident
under these stress conditions or an active mechanism of downregulation by such bacteria.
Moreover, further localization of NOD2 needs to be examined in the muscle of rainbow
trout. Bacterium, S. warneri, has been found to be located inctracellularly in epithelial, goblet,
and muscle cells. Currently, we are examining the role of NOD2 in sensing S. warneri in the
muscle of rainbow trout using FISH. Further analysis of expression of NOD2 in relation to S.
warneri in the muscle of rainbow trout needs to be examined, as initial observations indicate a
positive correlation.
Overall, this study establishes a framework for study Crohn’s disease related proteins,
using a comparative model, in order to ethically and effectively treat these patients. Due to the
morphology of trout NOD2, being orthologous to human NOD2, we can use rainbow trout to
understand the function of NOD2 in mucosal surfaces. Because CD is often affected by stress,
being able to understand NOD2 under stress conditions is imperative. This is why rainbow trout
is an ideal model organism because of the stress models we have established. In addition to the
stress models, the in vitro models allow us to analyze CD related proteins in relation to
commensal and pathogenic bacteria. This allows us to extrapolate information about these
symbiotic and dysbiotic relationships that is a crucial factor for treating CD patients. Rainbow
trout is an ideal organism to study CD, establishing a framework in which we can study CD in
dynamic ways, ultimately leading to a more effective treatment of CD.
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