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CHAPTER I: REPTILIAN IMMUNITY: THERE IS MORE THAN MEETS THE SCALE
1 │ OVERVIEW
Immunity is a mechanism that allows an organism to protect itself from potential pathogens. In
vertebrates, immune function has been well defined in mammals, but for non-model organisms
such as reptiles, much less is known. It has been noted several times in the literature that
characterizing immunity in non-model organisms can provide value to our overall
understanding of immunity. For instance, studies on the variable lymphocyte receptor (VLR)
genes of lamprey and hagfish, two jawless fishes, has revealed important clues about the
evolution of antigen recognition within jawed and jawless vertebrates (Boehm et al., 2012), and
the fruit fly, Drosophila melanogaster, now serves as a popular model organism to study
immune regulation at the level of a whole organism. Beyond this established role, D.
melanogaster also played an important role in understanding innate immunity with the
discovery of the Toll receptor/pathway, which would later provide important insight for Toll-like
receptors (TLRs) in mammals (Buchon, Silverman, & Cherry, 2014). Here I will review the
current knowledge of reptilian immunity, with special attention to our study system, the Red-
eared slider turtle (Trachemys scripta), and other chelonians. I also aim to provide relevant
examples of their contribution to the larger understanding of immunity and potential future
directions for reptilian immunity-oriented research.
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2 │ STUDY SYSTEM
Turtles are long-lived ectotherms that can survive in a variety of environmental conditions. T.
scripta is a species of pond turtle that is native from Illinois to the Gulf of Mexico. While they
are widely distributed, they all inhabit quiet freshwater areas with aquatic vegetation and areas
where they are able to bask (Ernst, Barbour, & Lovich, 2009). Females typically lay eggs during
the nesting season (May-September) by digging nests in dry unshaded areas near water (Cagle,
1946). Female sliders have been observed to lay 1-30 eggs (10.5 average) which have an
incubation period of 61.1-108 days (Ernst et al., 2009). As the female leaves the nest after
laying eggs, providing no post-oviposition parental care, these eggs are often subject to heavy
predation with a 10.5% survivorship rate from egg to hatchlings aged 1 year in the wild (Frazier,
Nat, Gibbons, Greene, & Frazer, 1990). Survivorship rates increase from 10.5% to 53.9% at age
1-2 and over 80% at all ages after that (Frazier et al., 1990). The incubation temperature of the
eggs is of particular importance as this species, like many other turtles, has temperature-
dependent sex determination (Janzen & Paukstis, 1991; Janzen & Phillips, 2006; Zimmerman,
Vogel, & Bowden, 2010), with warmer temperatures producing females. Upon hatching, they
are exposed to the microbe-rich soil. In more northerly populations, they overwinter in the nest
until early spring when warmer temperatures are common (Ultsch, 2006). After emerging in the
spring, the hatchlings disperse to the water and development continues.
Adult sliders typically spend a majority of their time submerged, emerging to bask periodically,
but have been observed to be able to remain underwater for extended periods of time during
winter hibernation (Ernst et al., 2009; Ultsch, 1989). Their aquatic habitat can range from
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microbe-rich filled marsh-like environments with large to slow moving streams. Adults have
been recorded to live past the age of 35 years old in the wild, but with ideal conditions, the
slider may be able to live even longer (Ernst et al., 2009). This ability to be long-lived in the wild
is not confined to just one species, but common amongst many in the chelonian order (Henry,
2003). In the three-toed box turtle (Terrapene carolina triunguis), gravid females aged over 60
years were observed to have negligible difference in physiological processes when compared to
much younger turtles (Miller, 2001). Furthermore, there have been multiple tortoises being
recorded to live over 150 years in age, such as Jonathan, an Aldabra giant tortoise
(Aldabrachelys gigantea), estimated to be over 180 years in age (Hollins, 2012; Kettle, 2014).
Turtles are a valuable group to study due to their unique physiology and capacity to be long-
lived in wild while seeming to be unaffected by typical effects of senescence such as decreased
reproductive frequency with age and maintained in conjunction with a type 3 survivorship
curve for most species (Congdon, Nagle, Kinney, & Van Loben Sels, 2001; Henry, 2003; Iverson,
1991; Miller, 2001). It has also been implicated in studies that the immune system of the turtle
may be unaffected by senescence as they seem to have little alteration in humoral immune
responses as they age. This was demonstrated through B cell function and antibody production
in 2013 study and also in a 2010 study which observed that temperature highly affects humoral
immune response, but age differences were not present (Zimmerman, Clairardin, et al., 2013;
Zimmerman, Carter, Bowden, & Vogel, 2017).
The reptilian immune system is commonly categorized into two main parts, the innate
component, and the adaptive component as seen in other vertebrates such as mammals. It is
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important to note that these components are not isolated, as they need one another to
function properly to produce an immune response. Pathogen clearance in the body, for
instance, typically starts when macrophages of innate immunity engulf foreign materials but
then present these internalized proteins to circulating T cells in order to mount adaptive
immune responses (Cannon & Swanson, 1992). In this way, the macrophage demonstrates
interconnectivity of the two components of the immune system. Reptilian species possess
many of the same lymphoid organs as mammalian species, such as a functioning thymus and
spleen, but lack structures such as lymph nodes and Peyer’s patches commonly seen in
mammals (Zimmerman, Vogel, & Bowden, 2010). Currently, six types of leukocytes have been
identified by morphology within turtles, these include basophils, eosinophils, lymphocytes,
monocytes, azurophils, and heterophils (Stacy, Alleman, & Sayler, 2011). Despite there being
much known about the physiology of turtles, and T. scripta in particular (Frazier, 1990), many
aspects of their immune system are not well defined. Within the last decade, however, there
have been several studies in T. scripta that have given insight into both their adaptive and
innate immunity (Palackdharry, Sadd, Vogel, & Bowden, 2017; Zimmerman, Clairardin, et al.,
2013; Zimmerman, Bowden, & Vogel, 2013; Zimmerman, Vogel, Edwards, & Bowden, 2010). It
has been reported that turtles, including T. scripta, contain populations of B cells with
phagocytic potential, not unlike that of the B-1 cell in mammals (Muñoz, Franco-Noguez,
Gonzalez-Ballesteros, Negrete-Philippe, & Flores-Romo, 2014; Zimmerman, Vogel, Edwards, et
al., 2010). Given that turtles appear to share some, but not all, immune functions in common
with other vertebrates, they provide an interesting group for further study.
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3 │ INNATE IMMUNITY
Innate immunity serves as the first line of defense against potential pathogens. Because of this,
innate immune responses are typically quick and non-specific. Innate immunity utilizes
physical/chemical barriers (e.g., epithelial skin layer), anti-microbial peptides (AMPs) (e.g., α-
defensins and β-defensins), the complement cascade, innate immune cell responses, and
signaling proteins (e.g., cytokines) until it clears the threat, or provides enough time for an
adaptive response to occur. The innate branch of the immune system is thought to have
evolved first between the two major branches, with the adaptive branch developing much
later. Researchers determined that innate components such as first TLR gene in multicellular
eukaryotes date back to roughly a billion years ago (Ausubel, 2005; Buchmann, 2014;
Kvennefors et al., 2010; Temperley, Berlin, Paton, Griffin, & Burt, 2008). While components of
adaptive immunity were first being observed roughly 500 million years ago in invertebrates
(Delvaeye & Conway, 2009; Iwanaga, 2002) and in ancient jawed fish around 430 million years
ago (Bayne, 2003).
The complement system consists of three main pathways: classical, alternative, and lectin,
which all utilize a suite of specific proteolytic enzymes to modify the abundant plasma protein,
C3. Modification of C3 results in fragmentation of the protein, where one fragment acts as an
opsonin, while the other fragment functions as an inflammation mediator (Merle, Church,
Fremeaux-Bacchi, & Roumenina, 2015). It is also important for B and T cell regulation within
immune responses (Dunkelberger & Song, 2010). In studies of immunodeficient patients and
mice with genetic knockouts, it’s been observed that compromise of the complement cascade
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results in increased S. pneumoniae and Haemophilus influenza prevalence, but also neisserial
infection and others as well (Dunkelberger & Song, 2010; Ross & Densen, 1984). Seeing that the
complement is so important mammals, it is no surprise that its presence has also been
observed in reptiles. In reptiles, it has been shown that they contain both the alternative and
classical complement pathways, with the optimum effectiveness of each pathway being
modulated by temperature (Koppenheffer, 1987). In a study using two different reptiles, the
Western fence lizard (Sceloporus occidentalis) and the Southern alligator lizard (Elgaria
multicarinata), it was observed that the bactericidal activity of the alternative complement
pathway is more aggressive than sera from humans and deer mice (Peromyscus maniculatus),
being able to kill the Lyme disease spirochete Borrelia burgdorferi (Kuo, Lane, & Giclas, 2000).
While a study in the Common map turtle (Graptemys geographica) and T. scripta have shown
that external temperatures effect on complement activity as well (Freedberg et al., 2008). A
recent study in the Prairie rattlesnake (Crotalus viridis) found that their serum had high
bactericidal effectiveness and was able to inhibit bacterial growth (Baker & Merchant, 2018).
Antimicrobial peptides are present in most living organisms and are typically less than 200
amino acids in length (Bulet, Stöcklin, & Menin, 2004). There is much diversity in the structure
of these peptides with linear α-helical amphipathic peptides being observed, while cyclic and
open-ended cyclic structures are also present in organisms. Of these differing structures, many
families of proteins have been characterized such as the defensin and cathelicidin families (van
Hoek, 2014). Most AMPs have cationic regions in the protein which allow them to interact with
the anionic cell walls and phospholipids layers of pathogens (Bulet et al., 2004). AMPs have
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been identified in four orders of reptiles: Testudines (turtles and tortoises), Sphenodontia
(tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilians) (van Hoek, 2014). In the
American alligator (Alligator mississippiensis), several AMPs have higher bactericidal qualities
than their mammalian counterparts (Merchant, Roche, Elsey, & Prudhomme, 2003), with a
recent study showing promising effectiveness against multidrug-resistant bacteria (Barksdale,
Hrifko, & van Hoek, 2017). In the Komodo dragon (Varanus komodoensis), 48 potential novel
cationic antimicrobial peptides (CAMPs) have been described, with one peptide having
significant potency against Pseudomonas aeruginosa (Bishop et al., 2017), another multidrug-
resistant bacteria (Driscoll, Brody, & Kollef, 2007). At this time, much effort is being put forth to
characterize novel CAMPs effective against particularly troublesome pathogens (i.e. multidrug-
resistant bacteria) against which modern medicine is losing the battle. In support of this, a
recent study in the Chinese alligator (Alligator sinensis) identified and characterized 20 novel β-
defensin genes, in an effort to assist with the investigation of their potential as an antibiotic
alternative (Tang, Wang, Wan, & Fang, 2018). Many of these studies focus on the Crocodilia and
Squamata clades, with few studies focusing on AMPs in turtles in the literature (Chattopadhyay
et al., 2006; Lakshminarayanan et al., 2008)
Regarding innate cellular responses, many innate immune cell types have been identified in
reptiles. For example, mammals contain neutrophils, which are short-lived granule-containing
phagocytes, while reptilian and avian species contain cells with similar functionality called
heterophils. Heterophils have nuclei with more a rounded appearance than an oval-like one,
typical of neutrophils (Campbell, 2004; Montali, 1988). Both cell types retain the innate
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functions of phagocytosis and the ability to release granules, and both represent a majority of
leukocytes in their respective taxa (Martins, Alevi, Azeredo-Oliveira, & Bonini-Domingos, 2016).
Interestingly, heterophil numbers have been shown to vary with seasonal temperature in
reptiles, with higher numbers found in the warmer seasons relative to cooler seasons, but in
mammals, this seasonal trend is absent, likely due to their endothermic nature (Troiano, Gould,
& Gould, 2008). In addition to heterophils, reptiles contain typical granulocytes (eosinophils,
basophils) and the azurophil, which is cytochemically and functionally similar to the monocyte
(Stacy et al., 2011). It has been observed in snakes however that their function is more
reminiscent of the neutrophil (Campbell, 2004). Reptiles also contain typical cells of the myeloid
lineage, such the monocyte, and macrophage (Campbell, 2004; Stacy et al., 2011), which are
also very important in innate and adaptive immune responses. Thus, while reptilian innate
immunity is functionally similar to mammalian innate immunity, differences in its effectiveness
and specific cellular components have given rise to many studies that further demonstrate the
need to better understand the reptile immune system.
4 │ ADAPTIVE IMMUNITY
A key component of adaptive immunity is the ability for organisms to form specific immune
responses to pathogens. This is accomplished by the generation of memory and non-memory
lymphocytes with genetically diverse antigen receptors (Cooper & Alder, 2006). In vertebrates,
adaptive immunity is composed of the humoral component and the cell-mediated component.
Adaptive immune cells develop from a common pluripotent hematopoeitic stem cell, which is
the precursor to all lymphoid, myeloid, and erythroid cell lines (Bryder, Rossi, & Weissman,
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2006). These stem cells are located in the red bone marrow of vertebrates, which is the primary
center of production of lymphoid cells and maturation center of the B cell. In mammals,
lymphoid structures such as the spleen and lymph nodes contain germinal centers which are
responsible for the activation of this cell.
4.1 │Cell-Mediated Immunity
The cell-mediated component of the adaptive immune system is mediated by a lymphoid cell
called the T cell. T cell progenitors are produced in red bone marrow and T cells undergo
maturation in the thymus. In mammals, T cells are necessary for B cell activation, responsible
for cell-mediated apoptosis when an intracellular infection arises, and also are shown to have
many regulatory functions (Mosmann & Sad, 1996). These responses are determined primarily
through the T cell receptor (TCR), major histocompatibility complex (MHC) receptor, as well as
other molecules (Rossjohn et al., 2015). T cells are typically classified by the types of co-
receptors expressed, which either give a “helper” function (CD4) or an effector function (CD8).
In reptiles, little is known about the function and development of T cells. Genes for CD4, CD8,
CD3, TCR, and MHC I and II were identified in the genome of the Painted turtle (Chrysemys
picta) (Bradley Shaffer et al., 2013). These receptors have also been identified in other reptiles
as well (Edwards, Grahn, & Potts, 1995; H. C. Miller, Belov, & Daugherty, 2005). In a study done
using the Carolina anole (Anolis carolinensis), the Siamese crocodile (C. siamensis), and A.
sinensis, the CD1 gene, a type of antigen-presenting molecule, was located; CD1 has a
significant role in T cell activation (Yang et al., 2015), providing further existence of their
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presence of T cells in reptiles. Many early studies involved observations of the size of the
thymus (Borysenko & Cooper, 1972). While thymic involution is observed to occur with aging in
mammalian species (Bodey et al, 1997), it’s been shown that turtles and other reptiles undergo
seasonal involution (Saad & Zapata, 1992). Involution in mammals leads to accumulation of
memory cells and decreased ability to respond to new infections (Cicin-Sain et al., 2007;
Mackall, Punt, Morgan, Farr, & Gress, 1998; Naylor et al., 2005). In the Caspian turtle
(Mauremys caspica), it was concluded that changes in the organ structure were linked with
seasonal changes (Leceta, Garrido, Torroba, & Zapata, 1989). This seasonal involution has been
shown to result in higher thymic cell numbers and response to chemotaxis in the summer and
decreased numbers in the other seasons (Muñoz & De la Fuente, 2001). More studies must be
done to understand the total effects that thymic involution trigger in reptilian species and
effects on memory cell production.
T cell functionality is thought to be very similar to that of mammalian counterparts, with
observed cell proliferation and chemotaxis when exposed to mitogens such as concanavalin A
(Con A) and phytohemagglutinin (PHA) in the Northern tuatara (Sphenodon punctatus) and M.
caspica (Burnham, Keall, Nelson, & Daugherty, 2005; Muñoz & De la Fuente, 2001). A CD3+ T
lymphocyte population has been isolated in the Green sea turtle (Chelonia mydas) (Muñoz et
al., 2009) and work done on understanding hormonal effects on cell-mediated responses in
separate experiments (Belliure, Smith, & Sorci, 2004; El Masri, Hakim Saar, Hesham Mansour, &
Badir, 1995). Beyond these few studies, literature on actual cell functionality in vitro is sparse,
and much more work needs to be done to truly understand T cell function and cell-mediated
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immunity in reptiles. With advancements in genomic studies, characterization of genes such as
those in the TLR family are the becoming the targets of study (Priyam, Tripathy, Rai, & Ghorai,
2018). Hopefully, future work will yield more reagents to determine whether differing
immunophenotypes exist within the reptilian system, such as separate T cell populations.
4.2 │ Humoral Immunity
The humoral arm of adaptive immunity allows organisms to have prolonged, specialized
immune responses to pathogens and is vital to protection at mucosal surfaces. The primary
lymphoid cell responsible for this type of immunity is the B cell. As mentioned earlier, its
progenitor is produced and matures in the bone marrow. The B cell’s primary purpose is to
produce antibodies that serve a variety of functions such as opsonization, neutralizing
pathogens, and promotion of complement activation (Sebina & Pepper, 2018). In mammals,
different B cell subsets exist, the main ones being the B-1 subset and the B-2 subset (Allman &
Pillai, 2008; F. Martin & Kearney, 2001). These two subsets differ in their function and
phenotype. B-2 cells are the main B cell population circulating in the blood and lymph of adult
mammals. They produce high affinity, specific antibodies, and maintain immunological
memory. In contrast, B-1 cells are located primarily in the peritoneal and pleural cavities in very
low numbers (Haas, 2015; Holodick & Rothstein, 2015). They also typically produce more
polyreactive, low-affinity antibodies (Griffin, Holodick, & Rothstein, 2011; F. Martin & Kearney,
2001).
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It is unknown if B cell subsets exist in reptiles, however, many lines of evidence suggest that B
cell function is more equivalent to mammalian B-1 cells. Characteristics of antibody production
in reptiles more closely resemble B-1 than B-2 cells. Following immunization, there is a modest
increase in antibody titer in the serum which happens slowly over time and titer does not
change significantly with a second exposure (T.M Work, Balazs, Rameyer, Chang, & Berestecky,
2000; Zimmerman, Vogel, & Bowden, 2010). Antibodies are polyreactive and commonly
referred to as natural antibodies (Panda & Ding, 2015), which characteristically have a low
binding affinity to multiple epitopes (Middleton, Nelson, Gartrell, & La Flamme, 2015;
Zimmerman, Bowden, et al., 2013). Mammalian B-1 cells have also been shown to produce
primarily natural antibodies (IgM, IgG3, and IgA), but lack production of higher affinity
antibodies (Ohdan et al., 2000). Genomic evidence and antibody analyses have shown that
reptiles contain IgM (Sun, Wei, Li, & Zhao, 2012), IgD (Gambón-Deza & Espinel, 2008; L. Li et al.,
2012; Wei et al., 2009; Xu, Wang, & Nie, 2009), two forms of IgY (L. Li et al., 2012; Wang et al.,
2012) and in the Leopard gecko (Eublepharis macularius), the presence of an IgA-like (Gambón-
Deza, Sánchez-Espinel, & Valdueza Beneitez, 2007). It is important to note that IgY has only
been shown to be expressed in avian and reptilian species (Warr, Magor, & Higgins, 1995;
Zhang, Calvert, Sutton, Doré, & Dor, 2017). IgY is functionally similar to IgG in mammalian
organisms (Wang et al., 2012), and thought to be the evolutionary precursor to IgG and IgE
(Pettinello & Dooley, 2014; Zhang et al., 2017). While one form of IgY retains the ability to bind
to Fc (Fragment crystallizable) receptors for leukocytes that express it, another truncated form
exists as well, with a hypothesized role in antigen neutralization without inflammation (L. Li et
al., 2012; Thierry M. Work et al., 2015). Fc receptors are expressed on leukocytes and the
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different types Fc (e.g. Fc-gamma) allow antibodies to bind and help with pathogen
identification which can induce cytokine release, engulfment of a pathogen, among other
functions (Raghavan & Bjorkman, 1996).
B-1 cells in mammals produce mainly low-affinity antibodies and usually do not participate in
germinal center reactions. B-2 cells are the cells that produce high-affinity antibodies and
interact with T cells in the germinal centers of lymph nodes. Notably, turtles lack lymph nodes
and germinal centers (Rios & Zimmerman, 2015). Antibodies produced by reptilian B cells are
generally thought to be low affinity from an experiment done in T. scripta. Using LPS
(Lipopolysaccharides) expressed from Salmonella, it was observed that while antibody
production was increased with age and season, the overall avidity of antibody to the antigen
was not altered (Zimmerman, Clairardin, et al., 2013). In a competitive binding ELISA using
keyhole limpet hemocyanin, overall binding avidity was not altered even with repeated
exposures to the antigen to simulate vaccination, whereas, in mammals, secondary doses of
antigens typically result in significantly higher recorded binding to antigens (Zimmerman,
Bowden, et al., 2013). Further, reptilian B cell populations more closely resemble mammalian B-
1 cells due to their ability to undergo phagocytosis (Gao et al., 2012; Novaes e Brito et al.,
2010). Following the discovery that some amphibian species and jawed fish contained B cells
capable of phagocytosis (J. Li et al., 2006; Øverland, Pettersen, Rønneseth, & Wergeland, 2010),
our lab was able to identify such cells in the turtle (Zimmerman, Vogel, Edwards, et al., 2010).
Phagocytic B cells were also identified in the Loggerhead sea turtle (Caretta caretta), giving rise
to the idea that these types of cells may be conserved in many other types of turtles (Rousselet
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et al., 2013). While researchers first identified phagocytic B cells in mammals in 1995 in mice (M
A Borrello & Phipps, 1995), there was relatively little work done on this previously
unappreciated B cell function. More recently, they have been discovered in human, non-human
primates, and many other mammals (Griffin et al., 2011; Haas, 2015), and they belong to the B-
1 subset. Thus, several lines of evidence suggest reptilian B cells are more similar to mammalian
B-1 cells.
Due to the presence of this cell in many species, many of which with less specific adaptive
immunity, it is possible that this B-1 cell could represent the primordial B cell, with it gaining
more effective humoral functionality as time progressed potentially through gene duplication
events giving rise to the B-2 subset (Popi, 2015). Some scientist predict that due its ability to
expresses cell surface protein such as F4/80 and Mac-1 when cultured in macrophage-like
conditions (Melinda A. Borrello & Phipps, 1996), that this bi-potential nature may be indicative
of a myeloid origin, while others have argued that due to their unique cell surface markers and
ability to produce antibodies indicate a clear lymphoid origin (Griffin & Rothstein, 2012). There
has also been an alternative hypothesis, the “induced-differentiation” hypothesis, states that
the B-1 cell could have always existed concurrently with the B-2 cell and formed from a
common ancestor (Berland & Wortis, 2002a). Similar occurrences of cells with bipotential
nature such as the CD1-restricted natural killer T cell have also been subject to study (Melinda
A. Borrello & Phipps, 1995, 1996). Regarding the evolution of the natural killer T cell, it is
thought to be a result of CD1 genes evolving rapidly and diverging substantially from the
reptiles within eutherian species (Kumar et al., 2017), which differ from both of the above
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hypothesizes of B-1 evolution. It is unknown whether a subset of these cells exist in reptiles, but
there has been discovery of the CD1 gene mentioned earlier. Overall, these B-1 cell findings
suggest immune subsets may represent more of a gradient of immunophenotype, as opposed
to a strict subset with no plasticity and this plasticity can be altered in specialized conditions
depending on the need of the organism. As it stands now, the evolutionary origin of the B cell is
still unknown, but as the progression of evolution becomes more accessible in through
molecular studies, a definite answer will be hopefully discovered in the future.
5 │ GALT IMMUNITY
The establishment of a well-defined gut microbiota and management is well understood to be a
key to management of gut microbes in humans and other organisms (R. Martin et al., 2010; Wu
& Wu, 2012). Gut-associated lymphoid tissue (GALT) is the largest lymphoid organ in the body
with a reported 1012 lymphocytes, and higher antibody production than all other non-GALT
lymphoid tissues (Brandtzaeg, 1989; Mayer, 2000). GALT immunity is essential for protection
against ingested pathogens, systemic pathogens circulating in the digestive tract, and
maintenance of gut flora in general. GALT immunity itself is just a portion of the much larger
mucosa-associated lymphoid tissues (MALT), which encompasses all ingestive tissue and
excretive tissue as a whole, as well other mucosa-lined tissue (Brandtzaeg, Kiyono, Pabst, &
Russell, 2008). Common types of GALT structures include the tonsils, Peyer’s Patches (PP),
mesenteric lymph nodes, isolated lymphoid follicles (ILFs), and other tissues. GALT immunity
functions by having a mucous barrier slow or prevent antigens from passing directly into the
circulating bloodstream of an organism. If the antigens breach the mucous layer and epithelial
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layer they interact with the lamina propia. The lamina propia, in most vertebrates, is loosely
organized lymphoid tissue connected to gut epithelial tissues that house various immune cells
(Mayer, 2000). Aggregations of lymphocytes (i.e. PPs and Mesenteric lymph nodes) are
constantly monitoring antigens caught in mucus that line local gut tissues. This allows increased
antibody production and proper inflammation reactions. While PP formation is driven
developmentally, ILF formation has been found to be induced through enteric stimulation and
diet (Colombo, Scalvenzi, Benlamara, & Pollet, 2015; Kiss et al., 2011; Lee et al., 2012;
McDonald, McDonough, & Newberry, 2005).
Virtually nothing is known about reptilian mucosal immunity. While reptiles don’t contain PPes
or tonsils and most don’t produce IgA, they still do contain GALT. There have been studies that
have identified a lamina propria region within their digestive tract as well as potential lymphoid
cells and aggregations. (Borysenko & Cooper, 1972; Zimmerman, Vogel, & Bowden, 2010). T.
scripta and many other freshwater reptiles are submerged for the majority of their lives in
pathogen-rich environments and constantly ingest these waters, but for the most part,
maintain good health. Even those that don’t contain life strategies that involve submersion in
water are known to carry highly pathogenic bacteria to mammals such as Salmonella and don’t
exhibit signs of health decline (Warwick, Lambiris, Westwood, & Steedman, 2001), which has
sparked interest into adaptive immunity in S. punctatus for instance (Middleton et al., 2015).
ILFs in mammals have been observed to act much like PPs in their ability to mount mucosal
immune responses increasing production of IgA in fecal samples when stimulated with an oral
inoculation of Salmonella typhimurium in mice (Lorenz & Newberry, 2004). In an experiment
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done with transgenic mice, it was shown that absence of PPs decreased antibody production,
but antibody production was compensated by other tissues such as the ILF (Yamamoto et al.,
2000). So the absence PPs in reptiles shouldn’t be thought of as a deficiency in GALT immunity,
but rather just a different strategy of gut immunity. I will expand upon the potential presence
of these tissues in a study covered in chapter 2.
6 │ CONCLUSION
While I mentioned that immunity-based research in reptiles is sparse when compared to other
organisms, I should also mention some of the other roadblocks that hinder this progress. With
the rise of genomic-based research, there simply are is not enough genomic information of
non-model organisms. This problem not only plagues reptile immunity-based research, but
research in other areas as well. A common approach to overcome this in our lab has been to
utilize avian species as templates, but differences in binding sites, expressed proteins, and
other issues have slowed progress. As technology advances and prices decrease for sequencers,
however, eventually more data will become available. There also is not a large push to develop
transgenic reptiles like those seen in mice and D. melanogaster. Many of the breakthroughs
that we have seen in immunology have come about precisely because of this ability to
manipulate the genome in these organisms. It should also be noted that advancements in
research of these model systems have also been accelerated due to their utility in the
development of modern medicine. Finally, a lack of reagents available to conduct many
experiments due to a lack of knowledge of cell surface markers and reasons listed above.
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The reptile immune system has a unique placement phylogenetically among vertebrates.
Reptiles first emerged about 300 million years ago, while other vertebrates such as mammals
and dinosaurs did not emerge until about 100 million years later (Buchmann, 2014). With a
shared common ancestor with mammals and birds, they give great insight into how immunity
may have specialized over time within amniotic vertebrates. While it is known that the reptilian
immune system contains both innate and adaptive compartments, an in-depth understanding
of these components to the level of other vertebrates such as mammals will require much
additional study.
Progress in understanding immunity in reptiles has proceeded notably slower, compared to
other model organisms, but important discoveries have come about, nonetheless. For instance,
there has been the discovery that not only do reptiles produce AMPs, but also their greater
effectiveness against pathogens could potentially yield therapeutic-related outcomes. We now
understand that the ectothermic nature of reptiles also has effectiveness on not only
metabolism, but also immune function and also that reptiles contain phagocytic B cells, which
have also been observed in early jawed vertebrates and mammals. These findings not only defy
the traditional humoral role of the B cell, but also give rise to the idea that this cell may have
had a less specialized role originally. While these advancements in our knowledge exist for this
particular adaptive immune cell, a comprehensive characterization of T cell phenotypes has yet
to be conducted. These few studies highlight the need to understand more about the reptile
immune system. I have raised more questions than I am able to answer as it seems there are
endless topics to research within the reptilian. Our lab and many others have attempted to do
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our part in showcasing this. It is my hope, however, that this brief review on reptile immunity
spikes interest in others for potential areas for future research.
20
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37
CHAPTER II: TRACHEMYS SCRIPTA HATCHLING EXPOSURE TO SALMONELLA ALTERS PRESENCE
OF INTESTINAL LYMPHOID AGGREGATIONS
ABSTRACT
Gut-associated lymphoid tissue (GALT) is vital for protection against ingested pathogens and
maintenance of normal gut microbiota. In mammals, gut mucosal immunity and the lymphoid
tissues associated with it, are well characterized, including isolated lymphoid follicles (ILFs) that
consist primarily of B cells, and are found throughout the small intestine. In mammals, the
formation of ILFs is not developmentally driven like other lymphoid tissues such as Peyer’s
Patches (PPs), but is rather dynamic and induced through antigenic stimulation and diet. In
reptiles, gut mucosal immunity is much less understood. Reptiles lack lymph nodes and PPs, but
whether they have ILF-like structures is unknown. In this study, we first set out to determine if
ILF-like structures were present in the red-eared slider turtle, Trachemys scripta. We identified
that there are B cell aggregates that are contained within the small intestine using paraffin-
embedded sections and whole-mount immunochemistry and a primary antibody to turtle
antibody light chains; these aggregates appear similar to ILFs in mammals. To determine if the
observed ILF-like structures were inducible, similar to those in mammals, we introduced an
enteric Salmonella species through oral gavage to hatchling turtles. Analysis of intestinal tissues
revealed that the presence of the B cell aggregates was much higher in distal sections than in
proximal sections, and that turtles exposed to Salmonella exhibited significantly more of these
aggregates, in general, when compared to those given sham treatments. These studies provide
38
novel information about gut immunity in non-mammalian vertebrates and provide the first
evidence for ILF-like structures in reptiles.
1 │ INTRODUCTION
1.1 │ Management of Gut Mucosal Immunity in Mammals
Gastrointestinal (GI) tissues are essential to nutrient absorption in all vertebrates, but also
serve as excellent areas for microbial colonization (Kato, Kawamoto, Maruya, & Fagarasan,
2014; Stevens & Hume, 1995). It has been estimated that roughly 90% of the total cells
contained within the human body are actually commensal microbiota that colonize the GI tract
(Savage, 1977). Because of this, constant monitoring by the immune system must be
maintained in these tissues to prevent otherwise benign interactions from becoming
pathogenic and to also prevent/limit pathogen establishment. Management of this colonization
occurs through lymphoid tissue lining the digestive system such as the spleen, mesenteric
lymph nodes, Peyer’s patches (PPs), and Isolated Lymphoid Follicles (ILFs). These structures
house innate and adaptive immune cells that survey the GI tract and are commonly termed gut-
associated lymphoid tissues (GALT) (Brandtzaeg, Kiyono, Pabst, & Russell, 2008; Hamada et al.,
2002; Stevens & Hume, 1995). PPs, in particular, are a major inductive site for B cell responses
and are the primary site of IgA production in the small intestine in mammals (Jung, Hugot, &
Barreau, 2010). Here B and T cells interact in germinal centers to produce high affinity, isotype
switched antibodies. Interestingly, in mice, rats, and rabbits that lack functional PPs there was
little effect on IgA production, indicating that other gut-associated lymphoid tissues may be
able to compensate (Heatley et al., 1981; Keren et al., 1978; Yamamoto et al., 2000). However,
39
removal of both mesenteric lymph nodes and PPs in mice results in severely decreased IgA
responses and altered lymphocyte distribution, demonstrating that this ability to compensate is
limited (Yamamoto et al., 2000).
In addition to PPs and mesenteric lymph nodes, lymphoid aggregates contained within the
digestive tract, termed ILFs, have recently gained attention due to their unique inducible nature
and protective qualities (Kiss et al., 2011; Lee et al., 2012). ILFs have been found in mammals
such as humans (Moghaddami, Cummins, & Mayrhofer, 1998), rabbits (Keren et al., 1978), mice
(Hamada et al., 2002), and Guinea pigs (Rosner & Keren, 1984); they have a composition that
differs from the “traditional” GALT tissues such as the PP or mesenteric lymph nodes. ILFs are
typically visually less distinguishable than PPs or mesenteric lymph nodes in the gut, being
much smaller in size, but more numerous (Hamada et al., 2002; Keren et al., 1978). They appear
earlier in development than PPs, and are primarily composed of IgA producing B cells (Hamada
et al., 2002). ILFs are formed from cryptopatches with RORγt+ lymphoid tissue inducer
precursor cells (Bouskra et al., 2008). These precursor cells are necessary to induce the
recruitment of B cells and other cells for cryptopatch formation, then through microbial
interactions, they are further induced to recruit more cells and form immature ILFs and then
mature ILFs (Lochner, 2011). It is important to note that, in mature ILFs have functioning
germinal centers, while immature ILFs and cryptopatches instead contain a large population of
B cells, dendritic cells, and small numbers of T cells amongst other cell types (Bouskra et al.,
2008). Unlike mesenteric lymph nodes and PPs, the distribution of ILFs can be altered in
40
response to changes in the surrounding microbial community to maintain homeostasis
(Brandtzaeg et al., 2008; Knoop & Newberry, 2012).
While these tissues are compensatory in nature, ILF-deficient mice are shown to have a 10-100
fold increase in commensal bacteria (Bouskra et al., 2008). Furthermore, ILF hyperplasia is
observed in Activation-induced cytidine deaminase-/- mice with 100-fold increases in anaerobic
bacteria within ILFs in the small intestines (Fagarasan, 2002; Knoop & Newberry, 2012). Upon
antibiotic treatment, the presence of ILFs in these animals was observed to be dramatically
decreased (Fagarasan, 2002). These results suggest that ILFs play a large role in maintaining
populations of enteric bacteria. Beyond their presence in mammals, ILFs have also been
described in amphibians (Ardavin, Zapata, Villena, & Solas, 1982; Marshall & Dixon, 1978).
1.2 │ Reptilian Gut Mucosal Immunity
While there is extensive knowledge about gut mucosal immunity in mammals, much work
needs to be done to understand mucosal immunity in reptilian systems. There are many
differences in the physiology of reptiles that are reflected in their management of systemic
immunity, but also gut mucosal immunity. Reptiles are vertebrate ectotherms that contain both
cellular and humoral immune compartments (Zimmerman, Vogel, & Bowden, 2010). They
possess primary lymphoid tissues such as bone marrow and a functioning thymus (Borysenko &
Cooper, 1972; Saad & Zapata, 1992; Zimmerman, Vogel, & Bowden, 2010), but they lack certain
secondary lymphoid tissues such as lymph nodes and PPs which are vital to mammalian
systems (Solas & Zapata, 1980; Zapata & Solas, 1979). It is also thought that they lack germinal
41
centers (Pitchappan, 1980), which play a very important role in isotype switching and affinity
maturation of antibody responses in mammals. Turtles have been shown to have the ability to
produce IgM, IgD, and IgY antibodies (Li et al., 2012; Pettinello & Dooley, 2014). Our lab has
also shown that turtles primarily produce natural antibodies (Zimmerman, Bowden, & Vogel,
2013), which are polyreactive, and they possess B cells with phagocytic functionality
(Zimmerman, Vogel, Edwards, & Bowden, 2010). Most previous studies on reptile immune
function have focused on systemic immunity, leaving important knowledge gaps in other
aspects of their immune response.
Turtles have long lifespans and live in pathogen-rich environments; it is very likely that they
have robust mucosal immunity in their gut, but virtually nothing is known about reptilian
mucosal immunity. Given that turtles lack IgA, PPs, and germinal centers, although some non-
chelonian reptiles have IgA-like genes, it is unclear how their microbiome is managed (Sun, Wei,
Li, & Zhao, 2012). GALT has not been extensively characterized in reptiles, with much of the
literature consisting of descriptive studies with simple histological stains that are not able to
identify specific lymphocyte populations. From these studies, it is reported that there are
possible lymphoid structures in the intestines of the Common snapping turtle (Chelydra
serpentina), with researchers noting that they observed diffuse aggregates that were composed
of small lymphocytes in the submucosa (Borysenko & Cooper, 1972). In a later comparative
study, similar lymphoid structures were reported in close association with the lamina propria
throughout the small and large intestine and in the cloaca in the Caspian turtle (Mauremys
capsica) (Zapata & Solas, 1979). They also observed similar structures in the Viperine water
42
snake (Natrix maura), the Algerian sand lizard (Psammodromus algirus), and in the Ocellated
skink (Chalcides occelatus) (Zapata and Solas, 1979). Thus, there is a possibility that reptiles
could rely on small immune aggregates like the ILFs of mammals.
We hypothesized that, due to lack of traditional GALT, mesenteric lymph nodes, and PPs, turtles
will rely heavily on B cell lymphoid aggregates (e.g., ILFs) in their intestines to regulate gut
immunity. We first sought to identify lymphocyte aggregations using histological analysis. We
were able to locate a structure reminiscent of a lymphoid aggregation. Like mammals, we
predicted that the introduction of a novel microbe would increase the presence of these
structures in the small intestine. In order to test this, we used T. scripta hatchlings to detect if
we could identify populations of B cell aggregates within their small intestines using
immunohistochemistry with a turtle light chain-specific mAb. We were able to successfully
identify numerous small lymphoid aggregates that are composed of B cells. We then tested
whether these aggregations had the ability to respond to changes within the gut
microenvironment by introducing Salmonella to a subset of hatchlings.
2 │MATERIALS AND METHODS
2.1 │ Egg and Hatchling Care
All animals and eggs involved in the studies below were obtained from Banner Marsh State Fish
and Wildlife Area in Canton, IL under an approved IACUC protocol. Eggs were collected
from freshly laid nests, or from gravid females that were collected from baited traps. Gravid
females were transported to the laboratory and induced to oviposit by oxytocin injection
43
(Ewert & Legler, 1978). Eggs were incubated in moist vermiculite (approximately -150 kPa) until
hatching. Once hatched, hatchlings were moved to individual containers and held for 8-10
weeks before experimental treatment. Hatchlings were then randomly assigned to either the
sham (Salmonella-) group (N=37) or the exposed (Salmonella+) group (N=39) (Figure 1).
2.2 │ Salmonella Exposure
Salmonella enteritidis (ATCC 13076) was grown to an OD of 0.3 (≈5x105 cells/ml) in LB broth the
night before inoculations. 1ml of these cells were concentrated via centrifugation, then
resuspended in 20µ of LB broth on the day of the inoculations. A 1ml feeding tube containing
20µl of Salmonella in LB broth or sterilized LB broth for the sham treatment was administered
to hatchlings via oral gavage. Water samples were collected from individual containers
immediately before inoculation to determine Salmonella presence pre-inoculation. Cups were
sterilized with a 10% bleach solution, rinsed thoroughly, and the oral inoculations were
administered. Hatchlings were held for 48 hours to allow for colonization to take place. Water
samples were taken after 48 hours to determine Salmonella presence, and hatchlings were
euthanized. Following euthanasia, intestinal contents were flushed using a butterfly needle
with 1x PBS, pH 7.4 and plated to determine if any Salmonella was present in the intestinal
content. Testing for Salmonella presence was determined using Difco™ XLT4 (Thermofisher)
selective agar.
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2.3 │Small Intestine Histology
Sections of hatchling small intestine were collected from two individuals and fixed in a 4%
paraformaldehyde solution for 24hrs, then stored in a sucrose solution. Intestinal sections were
then embedded in a paraffin wax cassette and sliced into 7µM sections using a microtome.
Sections were then stained using Hematoxylin and Eosin. Protocols for slices were adopted
from Fletcher and Wibbles (Fletcher & Wibbels, 2014). Sections were photographed using a
Leica DMRBE. Histological sections were analyzed using the image processing software suite Fiji
ImageJ v2.00 (http://fiji.sc/; Schindelin et al., 2012). Images were modified for correct white
balancing using a macro written by Vytas Bindokas; Oct 2006, Univ. of Chicago. Modified by
Patrice Mascalchi.
2.4 │ Whole-Mount Immunohistochemistry
After being flushed with PBS, intestinal tissue was prepared for immunohistochemistry. Two 1-2
cm sections were collected from each hatchling; one section was excised proximal to the yolk
sac and one section distal to the yolk sac. Intestinal sections were prepared using a modified
version of whole-mount immunohistochemistry according to a protocol previously used on
adult mice (McDonald & Newberry, 2007). Next, sections were split longitudinally and mounted
on Sylgard 184 Silicone epoxy (Dow Corning Corporation) in six-well tissue culture dishes
(humidity chambers) lumen side up. They were kept hydrated with room temperature 1x PBS,
then were washed three times for five minutes each while shaking at 600 rpm in warm (37°C)
Hanks balanced salt solution with 0.1M EDTA (HBSS-EDTA) to remove the surface epithelial
layer and expose the intestinal lumen.
45
The intestinal lumen was then washed with HBSS-EDTA using a 30cc syringe with a 23-gauge
needle to ensure complete removal of the epithelial cell layer. Sections were then shaken for 10
minutes in cold 1x PBS and fixed with a 4% paraformaldehyde solution in 1x PBS for one hour.
Following fixation, tissues were treated with 1% hydrogen peroxide in 1x PBS for 15 minutes to
inactivate any native endogenous peroxidases. Intestines were then blocked overnight in a
50mM Tris buffer (pH 7.2) supplemented with 150mM sodium chloride, 0.06% Triton-X 100,
and 0.1% bovine serum albumin (BSA) shaking at 4°C. Following incubation, intestines were
once again incubated overnight with a 1:500 dilution of HL673 mAb conjugated to biotin in
buffer solution. Next, intestines were washed three times for ten minutes each with 1%BSA-1x
PBS and incubated with streptavidin-horseradish peroxidase (SA-HRP) (BD Biosciences) diluted
1:1000 for one hour. Three more subsequent washes occurred, and then intestines were
treated with diaminobenzidine peroxidase substrate (Metal Enhanced DAB Substrate Kit,
ThermoFisher Scientific) for 15 minutes. Finally, the intestinal sections were washed twice with
dH2O to stop any reactions and returned to 1x PBS. Images were taken at (2.5X) magnification
using a Leica camera attached to a Leica dissecting scope.
2.5 │Spot Proportion Determination
Intestinal images were analyzed using the image processing software suite Fiji ImageJ v2.00
(Schindelin et al., 2012). A grid pattern was placed on the images 2.5X magnification on the
dissection scope. All squares within grids had a surface area of 0.1mm2. Grids containing small
intestine were marked and then area added. Then, grids containing spots (developed through
46
immunohistochemistry) were marked and added separately. The proportion of spots marked
compared to the entire intestine was then calculated. Images were also edited for white
balancing using the aforementioned method in ImageJ.
2.6 │Statistical Analysis
All statistical analyses were performed in R statistical software v3.3.3 (R Development Core
Team, 2016) and the lme4 package (Bates, Mächler, Bolker, & Walker, 2014). A generalized
mixed model with a binomial distribution was performed on the presence or absence of
Salmonella in water samples collected both before and after treatment, and additionally on the
proportion of small intestine grid areas with positive B-cell staining. For Salmonella presence in
water, a model was fitted with Salmonella treatment as fixed effect, and clutch as a random
effect. The presence or absence of B-cell staining within small intestine grids was analyzed with
treatment, location (proximal/distal), and season (early/late) as fixed effects, with clutch origin
and hatchling as random effects, to account for multiple measurements being taken from the
same individual. Maximal models including possible interactions between fixed effects were
simplified by sequentially eliminating non-significant terms through likelihood ratio tests (LRTs),
and best-fitting models were chosen based on the Akaike information criterion (AIC). For factor
level comparisons, the package lsmeans (Lenth, 2016) was used to extract predicted
probabilities for treatment levels from the best fitting models.
47
3 │ RESULTS
3.1 │Intestinal Histology Yielded ILF-like Structures
While early histological studies identified lymphocytes by morphology in these tissues
(Borysenko & Cooper, 1972; Solas & Zapata, 1980; Zapata & Solas, 1979), the distribution of
specific B cells is unknown. Previous studies have identified B cell aggregations within the small
intestine of reptiles (Zapata & Solas, 1979). Intestinal histology slices showed an aggregation of
cells with lymphocyte-like cell morphology and placement in the lamina propia (Figure 2-3).
There were no apparent afferent or efferent vessels, indicating that aggregate was formed
locally, as opposed to a PP with drainage to a local lymph node (Figure 2B and 2D). Slices with
these structures were not continuous in all sections of the intestine and were easily
distinguishable from a blood vessel (Figure 2A), which was observed to be continuous in all
sections. Our initial observation support that these structures are lymphoid aggregates, but this
method does not allow us to confirm what the specific cell types are present.
3.2 │ILF-Like Structures are more Abundant in the Distal Portion of the Intestine
With positive identification of lymphoid aggregates, we needed verification through other
means to determine cell phenotype. Proximal and distal intestinal sections from untreated
hatchling animals were found to contain B cell aggregations within the small intestine (Figure
4). To determine if the presence of these structures corresponded with those observed in
mammals, such that more ILFs appear distally as opposed to proximally, we determine spots
number on proximal and distal section of intestinal tissue. The proportion of spots contained
within the small intestine show a highly significant difference by location, with distal sections
48
containing a much higher proportion of spots compared to the proximal sections (Figure 5;
χ2=175.0, d.f. = 1, p < 0.0001).
3.3 │Prevalence of Salmonella Pre-inoculation
Before we could determine whether exposure to microbes in the gut would alter the presence
of the B cell aggregations, we had to identify if any of our animals were carriers of Salmonella
prior to our experimental exposure. We predicted natural carriage rates would be low due to
being reared under laboratory conditions versus their normal soil habitat. Pre-inoculation water
samples for both groups of hatchlings showed a low rate of natural Salmonella colonization as
expected (Figure 6). There was no significant difference in Salmonella prevalence among
individuals randomly assigned to the two groups (χ2= 11.5, d.f. = 1, p = 0.0007). Forty-eight
hours following inoculation, however, the number of individual water samples testing positive
increased significantly in the group that received the oral gavage containing Salmonella (Figure
6). Thus, it is likely that these animals were shedding bacteria in water samples. Due to
inconsistencies with intestinal flush procedures, that data was not included in our final analysis.
3.4 │Salmonella Exposure Increases ILF-like Aggregations
We examined intestinal sections for the presence of spots developed through whole-mount
immunohistochemistry. We observed a significant difference in the proportion of spots
contained within the distal intestine between our sham and exposed treatment groups.
Hatchings inoculated with Salmonella had a higher proportion of spots when compared to
those who were given the sham treatment (Figure 7; χ2=4.84, d.f. = 1, p = 0.028). These findings
49
indicate that exposure of hatchlings to Salmonella via oral gavage was successful, and also
indicates that the observed B cell aggregations found in the small intestine are responsive to
microbe presence.
4 │ DISCUSSION
Mucosal immunity and GALT structures in reptiles have received relatively little study compared
to those of mammals. In our study, we sought to determine if and how B cells were distributed
in the small intestine of the turtle, T. scripta. We hypothesized these cells play an important
role, similar to mammals, in maintaining commensal microbiota and protection from
pathogens. Given the lack of lymph nodes in reptiles, we predicted that the B cells would be
found in lymphoid follicle-like structures. We were able to identify lymphoid cell aggregations in
the small intestine histologically. Using a monoclonal antibody specific for turtle antibody light
chain proteins, we were able to clearly visualize small B cell clusters throughout the intestine.
We also wanted to determine if we could induce these structures, with the presence of a newly
acquired enteric microbe. It is important to note, that this is also the first study in mucosal
immunity in a hatchling turtle, which informs us that hatchlings as young as 8 weeks have
potential to develop B cell aggregations in the intestinal tract in response to gut microbes.
We observed that more B cell aggregates were present in hatchlings exposed to Salmonella
compared to our sham treated hatchings. Spots were also more numerous in distal sections of
intestines compared to the proximal sections. This finding is consistent with previous literature
using non-specific histological stains examining potential GALT structures and lymphocytes in
50
M. caspica, with more migrating lymphocytes and lymphoid tissue being observed as they
progressed through the small intestine (Duodenum<Jejunum<Ileum) (Solas & Zapata, 1980;
Zapata & Solas, 1979). These findings are also consistent with the increased ILF presence in
distal mouse intestines (Hamada et al., 2002; Lorenz, Chaplin, McDonald, McDonough, &
Newberry, 2003), suggesting that the distal portion of the small intestine is particularly
important for microbe management, as higher loads of bacteria have been recorded further in
the digestive tract (Donaldson, Lee, & Mazmanian, 2015). While these differences are
congruent with observations from other studies, explanation of the observed differences could
also be due to previous microbe exposure via the yolk. It is known that maternal yolk contains
antibodies and enzymes to support immunity of offspring in many organisms (Kovacs-Nolan &
Mine, 2012), but it is also known that egg yolk can contain pathogens (Parveen, Rahman,
Fakhruzzaman, Akter, & Islam, 2017), and it has been shown that microbial incubation in yolk
can also promote bacterial colonization in some cases. For instance, in a study examining
Salmonella Enteritidis infection in mice, scientists discovered that Salmonella grown in egg yolk
displayed greater illness, disease markers, and colonization than those grown in LB broth or
from mice previously infected with Salmonella (Moreau et al., 2016). The hatching yolk sac was
used as a marker to determine proximal and distal sections of the small intestine, with all
proximal sections being located proximally to the sac. Because of this, there was potential
differential pathogen exposure from the yolk sac, which may have also aided in the differences
observed in these B cell aggregations unintentionally, even though the entire intestinal section
should have received Salmonella via oral gavage for exposed individuals.
51
Pre-inoculation water sample testing for Salmonella showed that our hatchings contained a
very low prevalence of natural occurrence. Previous work by our laboratory and others did find
that hatchling turtles could acquire Salmonella from ingesting contaminated eggshells during
hatching (Holgersson, Nichols, Paitz, & Bowden, 2016), yet, infection did not persist more than
about 8 days (Pasmans, De Herdt, Dewulf, & Haesebrouck, 2002). Post-inoculation water
sampling showed a much higher prevalence of Salmonella in the exposed group. Ideally, water
samples containing shed microbes should serve as a proxy for bacterial colonization in the gut.
However, when the hatchlings were given Salmonella inoculations, Salmonella cells may have
coated the mouth or head of the hatchling upon introduction. While this was not ideal, it would
still promote ingestion of the pathogen, thus yielding the same result. We attempted to collect
intestinal flush samples to confirm colonization, but we experienced difficulty flushing intestinal
sections, so methods were changed mid-way through the experiment. Due to this, this data was
omitted from analysis, but it should be noted that a there was a low amount of growth
observed from flush samples collected. In the future, we hope to measure intestinal
colonization with a modified method that will be standardized across treatments. We also hope
to examine whether exposure to a more pathogenic enteric bacterium or immune stimulant
may cause altered presence of spots, as Salmonella is typically asymptomatic in healthy turtles
(Chiodini & Sundberg, 1981). It has been shown in mammals that methods of pathogen
recognition do not differ between commensal and pathogenic bacteria (Rakoff-Nahoum,
Paglino, Eslami-Varzaneh, Edberg, & Medzhitov, 2004), but methods for pathogen clearance
have been shown to be different (Haller et al., 2000). Salmonella is suggested to have
potentially have coevolved with reptilians from a more pathogenic organism to a commensal
52
organism (Bäumler, Tsolis, Ficht, & Adams, 1998), thus, exposure to a different pathogen may
yield different results. Would we see a larger inflammatory reaction from this exposure and
larger B cell recruitment as result? This question, among others, remains unknown.
These findings in combination with our post-inoculation water samples strongly suggest that
the B cell aggregates are able to respond to microbial presence. Their location is also consistent
with those observed in mammals and amphibians. It is of particular interest to know if T cells
are also present in the aggregates; however, no turtle-specific T cell reagents are currently
available. We also have only examined intestinal tissue in hatchling T. scripta, which
presumably would have a lower overall microbial load and less B cell development than in
adults. Future examination of these tissues in adults could provide even stronger evidence of
the ILF-like B cell aggregations.
5 │ CONCLUSION
To the best of our knowledge, this is the first study that has specifically identified B cell
aggregates within the intestine of a reptile using a primary antibody. We have also shown that
these aggregates are present in young hatchling turtles and that their number increases as you
progress distally in the small intestine. We have also shown that these aggregates have an
inducible nature. Hatchlings exposed to Salmonella increased the overall presence of these
aggregates when compared to those given a sham treatment containing no bacteria. It is our
thought that these aggregates seem to resemble isolated lymphoid follicles, which have similar
53
responses to microbes in other organisms. These important questions will be addressed in
future studies.
Fig. 1 A graphical representation of the experimental design using hatchling T. scripta in
our study.
54
Fig. 2 7µM sliced histological sections of hatchling small intestines stained with H&E.
Representative images are shown above all at 10X magnification. A) Intestinal
section with no lymphoid aggregation present; BV=Blood Vessel B) Another small
intestinal with visible lymphoid aggregations C) Lymphoid aggregation presence at
the base of villi D) Lymphoid aggregation presence within the lamina propia
55
Fig. 3 Oil immersion (100X) of intestinal section containing lymphoid aggregates. A) Small
round leukocyte-like cells within the base of the intestinal lumen B) Small round
leukocyte-like cells within the lamina propia C) Muscle cells shown within the
intestine D) Tip of villi with diffuse leukocytes contained within the interior section
surrounded by a single layer of columnar cells
56
Fig. 4 Clusters of B cells are present throughout hatchling intestines. Representative
intestinal images are shown. (A) Negative control intestinal section that received no
primary antibody (B) Proximal intestinal sections stained with primary antibody to
turtle light chains and (C) distal sections. Scale bars and magnifications are the same
for images (B) and (C).
57
Fig. 5 Frequency of B cell aggregates is increased in distal sections. Spot proportion was
determined as described for intestinal sections. Points represent model-estimated
probabilities. Distal Sections (n=45), Proximal Sections (n=41).
58
Fig. 6 Prevalence of Salmonella in tested water samples before and after inoculation. A
low proportion of individual’s water tested positive for Salmonella before treatment,
but a much higher proportion of animals tested positive for Salmonella shed in
water following inoculation of Salmonella by oral gavage treatment. Hatchlings were
randomly placed in sham (green bar) or exposure groups (orange bar) and water
samples collected and plated on selective media as described. After 48 hours, a post-
treatment water sample was tested. Bars represent model-estimated proportions.
Pre-treatment Sham Group (n=72), Pre-treatment Salmonella-exposed Group
(n=76), Post-Treatment Sham Group (n=64), Post-treatment Salmonella-exposed
Group (n=76).
59
Fig. 7 A higher proportion of spots were observed in hatchlings that were exposed to
Salmonella. Whole mount immunohistochemistry of the distal portion of hatchling
intestines was performed 48 hours after oral gavage. Images were analyzed as
described. Points represent model-estimated probabilities. Sham-Treatment Group
(n=38), Salmonella-exposed Group (n=48)
60
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CHAPTER III: CHARACTERIZATION OF THE PHAGOCYTIC CAPABILITIES OF THE T. SCRIPTA B CELL
ABSTRACT
The B cell is a crucial component of the adaptive immune response. Traditionally, it is thought
to function primarily for antibody production, but studies in our lab have identified a
population with phagocytic functionality in the red-eared slider (Trachemys scripta). Due to
many lines of evidence, we believe the phagocytic B cell in T. scripta may be very similar to the
B-1 cell in mammals. In this study, we aimed to determine whether their B cells had the ability
to recognize differing microbes, explore the size limitations of particle consumption, and
determine if these cells possess regulatory functionality as well. Unfortunately, the fluorescent
bioparticles that were used in our study were not compatible with our machinery and we were
not able to accommodate the many issues that arose during throughout our study. It is our
hope that in the future we will repeat this study, with new machinery and/or that our particles
compatible. We hope that this study will add more knowledge of reptilian immune cell
functionality and give more clues on the occurrence of phagocytic B cells in jawed vertebrates.
1 │ INTRODUCTION
1.1 │ Phagocytic B Cells In A Reptile
Adaptive immunity is essential for mounting specific immune responses by utilizing T and B
cells, while innate immunity is fast acting and relies on non-specific responses from innate cells
and molecules. Although B cells are typically associated with antibody production, several
subsets of B cells have been identified in mammals (Allman & Pillai, 2008). The major subsets of
68
mammal B cells are called B-1 cells and B-2 cells. B-2 cells can be thought of as “traditional” B
cells that produce many classes of antibodies. They also are located in several lymphoid organs
and respond strongly to protein antigens (Allman & Pillai, 2008). B-1 cells, however, have been
shown to primarily produce “natural antibodies”, which are also referred to as polyreactive
antibodies (Herzenberg et al., 1986; Panda & Ding, 2015). Polyreactive antibodies typically have
a low binding affinity to multiple epitopes and are usually of the IgM isotype, but natural forms
of IgA has been observed as well (Meyer-Bahlburg, 2015). B-1 cells in mammals are found in
niche populations mostly located in the pleural and peritoneal cavities and respond well to
carbohydrate antigens (Berland & Wortis, 2002b; Gao et al., 2012; L. Zhu et al., 2016).
While the immune system has been well studied in model species, less is known about
immunity in non-model species. Following the discovery that some amphibian species and
jawed fish contained B cells capable of phagocytosis (J. Li et al., 2006; Øverland et al., 2010),
our lab was able to identify such cells in the red-eared slider turtle (Trachemys scripta)
(Zimmerman, Vogel, Edwards, et al., 2010). The loggerhead turtle (Caretta caretta) and green
sea turtle (Chelonia mydas) have also been reported to contain phagocytic B cells, giving rise to
the idea that these types of cells may be conserved amongst most turtles and potentially all
reptiles (Rousselet et al., 2013; Q. Zhu et al., 2016). This innate phagocytic functionality is not
exclusive to the turtle however and has been subsequently characterized in many mammals
such as mice (Gao et al., 2012), humans (Souwer et al., 2009), and non-human primates (Haas,
2015).
69
It is unknown if B cell subsets exist in reptiles, however, several lines of evidence suggest that
their B cell function is more equivalent to mammal B-1 cells. First, characteristics of antibody
production in reptiles more closely resemble B-1 than B-2 cells. Following immunization, there
is a modest increase in antibody titer in the serum which happens slowly over time and titer
does not change significantly with a second exposure (T.M Work et al., 2000; Zimmerman,
Vogel, & Bowden, 2010). Generally, the antibodies produced are natural antibodies of low
affinity (Zimmerman, Bowden, et al., 2013).
Second, reptilian B cells possess phagocytic capability, which is only observed in the B-1
phenotype in mammals (Parra et al., 2012; Popi, 2015). Preliminary data from our lab suggests
that reptilian B cells can phagocytose a large number of inert 1um polystyrene beads
(Palackdharry et al., 2017), but how they target phagocytosis is unknown. It’s been shown that
B-1 cells utilize the B cell receptor for specification in mammals (Gao et al., 2012), but toll-like
receptor (TLR) recognition is known to play a role in the maturation process and potentially
recognition as well (Meyer-Bahlburg & Rawlings, 2012). Reptilian B cells produce a B cell
receptor and sequence data from the painted turtle suggests a variety of TLR genes are present
as well (Bradley Shaffer et al., 2013).
Finally, there is potential evidence, that the reptile B cell has regulatory functionality. B-1 cells
in mammals have been observed to play a role in inflammation and a subset of B-1 cells have
been aptly named, B regulatory cells due to this (Aziz, Holodick, Rothstein, & Wang, 2015). It
was observed in preliminary findings, that our B cells seemed to be more aggressive at
70
consuming polystyrene beads than the co-incubated adherent cells (i.e.
macrophages/monocytes)(Marrochello, 2016). It is known that B regulatory cells possess the
ability to secrete IL-10 which decreases the phagocytic ability of macrophages in-vitro (Popi,
Lopes, & Mariano, 2004).
In order to learn more about these unique cells, we sought out to answer some of the
questions mentioned above using T. scripta. First, we wanted to determine if the reptile B cell
has the ability to recognize different kinds of microbes. Thus, we modified our phagocytic assay
to use fluorescent microbes instead of beads as targets. Unfortunately, due to the extreme
brightness of the beads, we were unable to distinguish B and non-B cells in the assays. Future
studies might resolve the issue by using a fluorochrome to detect B cell staining that is much
longer in wavelength than FITC to prevent fluorescent bleed over.
2 │MATERIALS AND METHODS
2.1 │ Lymphocyte Collection and Preparation
During the nesting season, female adult T. scripta were collected from Banner Marsh (Canton,
IL) as approved by the IACUC. A 1:1 ratio of 3-4ml of pooled whole blood was collected and
added to RPMI 1640 (Life Technologies) and 1:30 of 0.1M ethylenediaminetetraacetic acid
(EDTA; Fisher Scientific) in a 15ml tube. Mixtures were then be carefully layered on a 50%
Percoll (MP Biomedicals, LLC) and saline solution with the final result being a 1:1 ratio of
blood/RPMI to Percoll/saline. Tubes were then centrifuged at 400xg at 4°C for 5mins without a
brake. The resulting buffy coat layer containing leukocytes was extracted and washed with
RPMI. A 15ml tube filled RPMI was then centrifuged for 5mins at 1500xg at 4°C with a brake
71
applied. RPMI was then decanted and resulting pellet was resuspended in 500µl of RPMI and
counted. Ideal cell concentration should be 6-7 x 106 cells/ml. One ml of resuspended solution
was divided into different tubes corresponding to the specific experiment (see below).
2.2 │Phagocytic B cell Assay
In vitro phagocytic assays were carried out as described in L. M. Zimmerman et al., (2013b).
Isolated leukocytes were adjusted to at least 5x105 cell/well and incubated with fluorescent
bioparticles (20:1), based on another experiment using similar bioparticles (Nuutila & Lilius,
2005) and previous experiments with FITC beads (Fluoresbrite Plain Yellow Green
Microspheres, Polysciences) (Zimmerman, Vogel, Edwards, et al., 2010) for control samples.
Cells and particles were incubated in 6 well plates at 29-30°C and 5% CO2 for 3 hours. The cells
and bioparticles were collected from the wells by vigorous pipetting and transferred to a
collection tube, filled with RPMI and washed for 1500 ×g for 5 min at 4 °C. The cells were
resuspended in 2ml of 1x Hanks’ Balanced Salt Solution (HBSS, Life Technologies)-1% BSA. In
order to wash away non-consumed beads, the beads/bioparticles and cells were layered 1:1 on
a cushion of 3%-4.5% Dextrose-Phosphate buffed saline. Following supernatant removal (which
contained the free beads), pelleted cells were washed and centrifuged as previously mentioned
and resuspended in 400μL 1× Hanks-0.5%-BSA. They were washed again and decanted until
only 50µl remain in the tube. A 1:10 ratio of normal rat serum was added to block non-specific
binding and 1µl of biotin-labeled anti-turtle light chain monoclonal antibody (HL673 mAb,
University of Florida Hybridoma Facility) was incubated for 15mins and the cells were stored on
ice. Cells were washed and then stained with 20µl of Streptavidin-spectral red (Southern
72
Biotech, 0.5mg/ml) diluted 1:1000 in 1× Hanks-0.5%-BSA and incubated in the dark on ice for
15min. They were washed again and 300µl of 1x HBSS-1% BSA was added to prepare the
sample for Flow cytometry. In order to avoid high background from the bioparticles, cells were
quenched with 200 µl of 1.2 mg/ml of Trypan Blue (MP Biomedicals) prior to running (Nuutila &
Lilius, 2005). Cells were analyzed immediately on a Becton Dickinson FACSCalibur flow
cytometer. A minimum of 10,000 events were collected and data was analyzed using CellQuest
Pro software (BD Biosciences).
2.3 │Bioparticles and Beads
There were three experimental groups: (Bioparticles from E. coli only, S. aureus only, and S.
cerevisiae only (Zymosan) that were all FITC-labeled. As a control FITC-labeled (0.5µm)
polystyrene beads were used since turtle B cells have previously been shown to phagocytose
these particles (Zimmerman, Vogel, Edwards, et al., 2010). Fluorescent bacteria and yeast
bioparticles were purchased from ThermoFisher Scientific. Five replicates were attempted per
group for statistical comparisons. Experiments were completed in groups. For example,
experiment 1 was completed using one blood pool sample with every experimental group at
once. A total of five different blood pools were used on five separate days.
2.4 │Microscopy
We utilized a Leica TCS SP2 for confocal microscopy to locate B cells that consumed
particles/beads by spotting them on a microscope slide following the phagocytic assay. We also
used a Leica DMRBE equipped with darkfield, DIC, and phase contrast as well as fluorescence
filter sets for UV, blue, green and GFP excitation to attempt to locate these cells as well. We
73
replaced the Streptavidin-spectral red stain for Goat anti-Mouse IgG (H+L) Highly Cross-
Adsorbed Secondary Antibody, Alexa Fluor 633 and 568 (Thermofisher), as our original
filter/laser setup on these microscopes were unable to locate the cells.
3 │ RESULTS
3.1 │Bioparticles were too Bright for the BD FACSCalibur
Previous research in our lab developed an assay to examine B cell phagocytosis using
polystyrene beads (Zimmerman, Vogel, Edwards, et al., 2010). Because different receptors,
such as TLR, may be involved in recognition of actual pathogens, we predicted phagocytosis of
pathogens may be more efficient than inert beads. Thus, we modified our phagocytic assay to
include Bioparticles obtained from Thermofisher. We selected bioparticles from the major
different types of microbes: E. coli for Gram-negative bacteria, S. aureus for Gram-positive
bacteria, and S. cerevisiae for yeast. In Figure 8, a typical scatter plot with lymphocyte gating is
shown (upper left panel). Staining with HL673 reveals specific B cell staining versus control (no
fluorochrome; top middle and right panels). Bottom panels show polystyrene beads alone.
Scatter plot on left, normal beads in middle and beads quenched by addition of trypan blue on
right.
A phagocytic assay was run with beads and bioparticles as described in methods. In Figure 9,
scatter, control staining, and B cell staining are shown for cells that did not receive beads (top
panels). Middle and bottom panels show cell populations following the phagocytic assay with
the indicated targets. In the bead sample, the upper left quadrant is showing an unusually high
74
level of B cell staining. Although in the upper right panel we can clearly distinguish phagocytic
FITC+ cells, it is not clear they are B cells in this experiment. Likely, too much SA-SR dye was
added causing a high background. For the bioparticles, for E. coli and S. aureus, there is also
evidence of a phagocytic population but for the yeast particles, the fluorescent was too intense
to distinguish from background.
Based on other reports (Nuutila & Lilius, 2005), we attempted to use Trypan Blue to quench the
bright signal. However, the resulted in all cells appearing false positive for the SA-SR dye
(Figure 10). Again, we were able to detect phagocytosis in 3 of the 4 samples (not in the yeast
sample) but were unable to determine if the cells were truly B cells. Figure 11 illustrates the
bioparticles themselves in trypan blue and even with maximal compensation settings the FITC
signal was being detected in the FL3 photomultiplier tube. Thus, we were unable to prevent
bleed over of the fluorescence into the photomultiplier tube where we collected the B cell
signal, and were unable to discriminate phagocytic B and non-B cells.
3.2 │Determination of Phagocytosis by Fluorescent and Confocal Microscopy
Due to our difficulty in obtaining data through flow cytometry, we tried to visualize if our
bioparticles were ingested by both fluorescent (Figure 12 and 13) and confocal microscopy. We
encountered difficulty locating a single B cell that underwent phagocytosis. Usually, ~2-5
percent of B cells undergo phagocytosis in T. scripta (Figure 9, Zimmerman et al., 2010b), so
visualizing unpurified populations of cells provided a very low probability of occurrence. We
also experienced high cell death, which could be due to slide preparation, as we did not fix our
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cells prior to staining. Fluorescent microscopy showed that FITC-coated particles were so bright,
they were visible in both GFP and RFP filters (Figure 13), as was the case for the flow cytometry.
We should note that quenching minimally altered the fluorescence for the scope as well.
Confocal microscopic analysis showed us a single potential phagocytic B cell. It was very
misshaped, however, and we couldn’t be sure if it was an actual B cell. Staining with secondary
Alexa 568 and 633 antibodies also didn’t show many positive-stained B cells (Figure 12). Thus
we were unable to confirm the B cells were ingesting the bioparticles.
4 │ DISCUSSION
Innate immunity is thought to have evolved prior to adaptive immunity (Buchmann, 2014;
Hirano, Das, Guo, & Cooper, 2011). Many vertebrates have populations of innate immune cells
with specialized innate functionality, such as macrophages and neutrophils. Thus, it is of
interest why innate immune function might have been retained in some cells of the adaptive
immune system, such as the phagocytic B cell identified in T. scripta. Understanding how and
why these cells contain this innate functionality provides novel information into the evolution
of the immune system. At first glance, it would seem that in a system where professional
phagocytes currently exist, this redundant function is unnecessary. Because of this, there’s
been much debate of the origin of the B-1 cell in the mammal, with some scientist conceding to
this idea that the B-1 cell could be of the myeloid lineage (Popi, 2015). As studies have
progressed, however, the literature has now uncovered the importance of these cells for
managing mucosal immunity by the production of IgA (Meyer-Bahlburg, 2015) among other
functions such as antigen presentation (Popi, Longo-Maugéri, & Mariano, 2016). Examination of
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these cells begs the evolutionary question, are B-1 cells artifacts from innate immunity that
specialized to become B-2 cells as the adaptive immune system evolved better microbe
recognition strategies or are these cells simply another cell subset? Evidence that these cells
are contained within reptiles, amphibians and early jawed fish (J. Li et al., 2006) provide
important clues to aid in resolving this argument.
It is unknown whether or not the B cells within T. scripta have the ability to recognize and
consume different types of microbes or are specialized in their phagocytic targets. Prior
phagocytic studies within turtles have only examined the uptake of phagocytic beads, but we
suspect pattern recognition receptors for microbial components may also play a role in this
process. Particle uptake in professional phagocytes has also been shown to decrease as the size
of the target increases in many different studies (Cannon & Swanson, 1992). If the size of the
target becomes too large, the B cell may attempt phagocytosis but fail (“frustrated
phagocytosis”), or it may switch to produce antibodies instead. As of now, no study has sought
out to see the size limitation of phagocytosis in phagocytic B cells or if the cells switch to
antibody production in T. scripta. Thus, we’d propose to investigate this in future studies using
beads and particles of different sizes.
It is currently not known if the B cell has antigen presentation capability following phagocytosis.
It has been reported in the literature in mice that both B-1 and B-2 cells have the capability to
also aid in antigen presentation (Lee-Chang et al., 2016; Popi et al., 2016). A future goal in our
lab is to determine whether or not this ability is retained within the reptilian B cell. A recent
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study was done in the turbot (Scophthalmus maximus) that determined that teleosts B cells rely
heavily on micropinocytosis for particle uptake (Y. Li, Sun, & Li, 2018). It remains an open
question if the reptile B cell utilizes these same mechanisms and future studies aim to explore
this as well.
5 │ CONCLUSIONS
We originally aimed to learn more about the phagocytic functionality within the B cell in T.
scripta in the 2017 nesting season. Unfortunately, due to many technical issues, this study was
unable to be completed. The various bioparticles we chose to use proved to be much more
fluorescent than we expected. Despite many efforts to work around this issue by quenching,
altering compensation/voltages in flow cytometry, and utilizing fluorescent and confocal
microscopy, we were still unsuccessful in relevant data collection. It is our hope in the future to
reattempt the proposed experiments and learn more about the phagocytic B cell in the reptile.
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Fig. 8 Sample flow cytometry analysis showing gating of cell/bead populations. Top left:
Scatter plots with forward scatter (FSC) and side scatter (SSC) showing gating of
lymphocyte populations. Top middle: Cells that did not receive SA-SR. Top right:
cells stained with HL673. Bottom left: scatter plot showing beads alone. Bottom
middle: fluorescence of beads only and bottom right: beads quenched with trypan
blue. Representative histograms from an individual blood pool are shown.
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Fig. 9 Turtle blood cells were able to phagocytose bioparticles, but fluorescence bleed over
prohibited identification of B cells. Top left: scatter plot of the blood sample. Top
middle; Control staining without SA-SR. Top right; Stained cells without beads
stained for B cells. Middle and bottom panels: Cells incubated with various
indicated target particles and stained with HL673-SA-SR. Representative histograms
from an individual blood pool are shown.
Scatter
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Fig. 10 Trypan blue failed to quench fluorescence bleed over. Samples were prepared as in
Figure 9 and trypan blue was added immediately prior to analysis.
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Fig. 11 Bioparticles quenched without cells still prove to be too bright. Bioparticles were
incubated directly in trypan blue and analyzed as in Figures 9 and 10
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Fig. 12 Fluorescent microscopy failed to detect stained lymphocytes. Following the
phagocytic assay with various bioparticles, samples were spotted onto a microscope
slide and viewed under the confocal microscope A) Bright field image of a potential
lymphocyte and a structure of unknown origin B) Same field of view as A but using
the RFP filter.
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Fig. 13 Bioparticles show false positive in GFP and RFP filters. A) Brightfield image with an
arrow indicating a blood cell and bioparticles in the background. B) A GFP-Filter
indicating E. coli particles clumped together with no interaction with blood cell. C)
An RFP-Filter with arrows indicating that bioparticles show false positive in this filter.
