The Effects of Digestion on Innate Immunity" (2015)

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University Honors Program Theses

2015 The ffecE ts of Digestion on Innate Immunity Rachel L. Luoma Georgia Southern University

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This thesis (open access) is brought to you for free and open access by Digital Commons@Georgia Southern. It has been accepted for inclusion in University Honors Program Theses by an authorized administrator of Digital Commons@Georgia Southern. For more information, please contact [email protected]. The effects of digestion on innate immunity An Honors Thesis submitted in partial fulfillment of the requirements for Honors in the Biology Department By Rachel L. Luoma Under the mentorship of Dr. Zachary R. Stahlschmidt Abstract Following a meal, an animal can exhibit dramatic shifts in its physiology that can result in rapid growth of the gut and heart, as well as a massive (>40-fold) increase in metabolic rate associated with the energetic costs of processing the meal. However, little is known about the effects of digestion on an important physiological trait: immune function. Thus, I tested the following competing hypotheses. First, digesting animals may upregulate their immune systems due to increased microbial exposure from ingested food. This hypothesis predicts that animals will exhibit greater immune function during digestion. Second, digesting animals may downregulate their immune systems to devote energy to the breakdown of food. This hypothesis predicts that animals will exhibit lower immune function during digestion. I tested my hypotheses by measuring two assays of innate immunity (the chief mechanism of host defense across taxa) on the blood plasma of corn snakes ( Pantherophis guttatus ) during and after meal digestion. I specifically measured hemoagglutination (antibody-mediated agglutination of foreign red blood cells) and hemolysis (complement-mediated lysis of foreign red blood cells) at two time points (1 and 7 day[s] post-feeding [dpf] to reflect digestive and nondigestive states, respectively). Hemoagglutination was higher at 1 dpf in support of my first hypothesis. Furthermore, females had higher lysis than males. In sum, I demonstrate the effects of digestive state on innate immune function, further highlighting the myriad physiological responses that occur in response to food intake and digestion.

Thesis Mentor: ______Dr. Zachary R. Stahlschmidt

Honors Director: ______Dr. Steven Engel

April 2015 Biology Department University Honors Program Georgia Southern University Introduction

Although animals must ingest and digest food to survive, they incur substantial

energetic costs when processing their food (Secor, 2009). This energy

expenditure, characterized as increased metabolic rate and heat production, is

called specific dynamic action (SDA). The SDA response varies in magnitude

(increase in metabolic rate of 25% to over 4000%) and duration (hours to weeks)

depending on the taxon of animal and the size of the meal (Secor, 2009). The

postprandial (postfeeding) metabolic response is caused by an upregulation in

digestive processes, characterized as increase in gut size and enzymatic activity

in addition to other physiological and morphological changes (Secor, 2009).

The immune system is a crucial physiological process for an organism’s survival,

yet the effects of digestion on the system’s function have been understudied. The

immune system is essential for the regulation of an organism’s interactions with

microbes (Ponton et al., 2013), and it is energetically costly to maintain and

upregulate (Martin et al., 2003). Innate immunity is the first line of defense

against foreign microbes (including those found on food) once they have invaded

the body (Matson et al., 2005), and it is also the chief form of immunity across all

taxa (Litman et al., 2005). Thus, innate immunity may exhibit a response to

digestion that is taxonomically widespread. Although, some studies have

examined the effects of nutrition (nutrient composition: Cotter et al., 2011; Moret

& Schmid-Hempel, 2000; food limitation: Kristan, 2007; hydration: Moeller et al.,

2013) on the innate immune system, the effects of digestion itself on the innate

immune system are unknown.

I used the corn snake (Pantherophis guttatus , an infrequent eater with a large

SDA response: Crocker-Buta & Secor, 2013) to examine the effects of digestive

state on two immune metrics (hemoagglutination and hemolysis) by testing two

hypotheses. My first hypothesis posits that digesting animals upregulate their

immune systems due to the increased microbial exposure from ingested food

(Conway, 1997). This hypothesis predicts that digestion leads to greater immune

function—that is, an upregulation in the immune system contributes to the SDA

response. Alternatively, my second hypothesis posits that digesting animals may

downregulate their immune systems to allocate energy to the breakdown of food.

This hypothesis predicts that digestion leads to lowered immune function. By

testing these hypotheses, I will demonstrate whether the innate immune system

is affected by the digestive state of an organism—specifically, whether energy is

allocated toward or away from innate immune function during digestion. This

study will elucidate a potential tradeoff between digestion and immunity, as well

as the further clarify where energy is allocated during the SDA response in

animals.

Methods

Study species and husbandry

Corn snakes are non-venomous, medium-sized, and native to the southeastern

United States (Gibbons & Dorcas, 2005). A captive sample of 32 corn snakes , aged 14-16 months were used to address my hypotheses. Snakes were the

offspring (1 st -3rd generation) of wild snakes caught in Beaufort County, South

Carolina, USA. Snakes were kept individually in translucent plastic enclosures

(27 cm x 41 cm x 15 cm) in a room with a 12:12 light:dark cycle. Enclosures had

subsurface heating elements that allowed snakes to thermoregulate along a

gradient of temperatures from 24.5-33°C. This thermal gradient encompasses

the preferred temperature range for corn snakes, 27-29°C (Roark & Dorcas,

2000). Snakes had ad libitum access to water and were offered food

(frozen/thawed adult mice that were 15-20% of weight of the snakes to which

they were offered) every two weeks throughout the study.

Experimental design

Over the course of the 8-week study, each snake’s blood was sampled at two

time points: 1 and 7 day(s) post-feeding (dpf). The order of the sampling was

randomized, and the duration between sampling was two weeks. Snakes were

sampled at these times points as their metabolic rate peaks 1 dpf and declines

back to pre-feeding levels approximately 4 dpf when fed 15-20% of their body

mass (Crocker-Buta & Secor, 2014). Thus, 1 dpf denotes the digestive state

while 7 dpf denoted the nondigestive state. During both sampling points,

intracardiac blood draws (0.3 ml) were made. Blood samples were placed on ice

prior to centrifugation at 5000 rpm for 5 min. Plasma was removed, and an

aliquot of 35 µl of plasma was stored at -80°C prior to immune assays (see below). All procedures were approved by the Institutional Animal Care and Use

Committee at Georgia Southern University (protocol #: I14004).

Hemoagglutination-hemolysis assays

Hemoagglutination and hemolysis assays were performed using the assay technique from Butler et al. (2013). Each plasma sample was serially diluted with

1xPBS (phosphate buffered saline) in a 96-well clear u-bottom plate. Sheep blood diluted to 1% was then added to each well, leaving the twelfth column as a control. Plates were incubated at 26.5°C, which reflects the preferred temperature of corn snakes (Roark & Dorcas, 2000; Stahlschmidt et al., in review) and scanned for hemoagglutination (Fig. 1). Hemoagglutination is characterized by natural antibodies, a type of immunoglobulin, that function as recognition molecules (Matson et al., 2005). Natural antibodies have the ability to opsonize foreign microbes causing agglutination, which in turn promotes the onset of the complement enzyme cascade (Matson et al., 2005). The plates were subsequently left at room temperature and then scanned for hemolysis (Fig. 1).

Hemolysis is characterized by complement, which can be activated by means of three different pathways that all lead to lysis of the foreign body (Trouw & Daha,

2011). Plates were independently and blindly scored by RLL and ZRS. Because our scores were highly correlated, I used the mean value of our scores to perform statistical analyses (see below).

High Plasma Concentration Low Plasma Concentration Titer = 1 2 3 4 5 6 7 8 9 10 11 Neg. Control

Result = Ly Ly Ly Ly Ag No No No No No No No Ly = Lysis Ag = Agglutination No = No lysis or agglutination

Figure 1. An example scan of a plate from the hemoagglutination-hemolysis assay performed on plasma obtained from corn snakes. Titers 1-4 show hemolysis. Titer 5 shows hemoagglutination. The negative control (PBS only) as well as the titers 6-11 illustrate a lack of either hemoagglutination or hemolysis. This example scan of a plate was scored lysis=4 and agglutination=5.

Statistical analyses

To determine the effects of digestion and sex on hemoagglutination and

hemolysis, t-tests were performed and two-tailed significance was determined at

p<0.05. Paired t-tests were performed to determine the effects of digestive state

because each snake’s immune function was determined at 1 and 7 dpf. Unpaired

t-tests were performed on male and female samples to determine the effects of

sex on immune assays. F-tests were performed prior to unpaired t-tests to

determine equality of variance. All data met the assumptions of parametric

statistics.

Results

Hemoagglutination was affected by digestive state—specifically, it was higher at

1 dpf than at 7 dpf for combined male and female samples (p=0.004, t 28 =3.2)

(Fig. 2). However, sex did not have an effect on hemoagglutination at 1 dpf

(p=0.48, t 27 =0.71) or at 7 dpf (p=0.35, t27 =0.96) (Fig. 2). Sex did not have a

significant effect on hemoagglutination overall either (1 dpf and 7 dpf combined)

(p=0.261, t 56 =1.1). Hemolysis was not affected by digestive state for combined

male and female samples (p=0.40, t 29 =0.86) (Fig. 3). However, sex did

significantly affect hemolysis overall (1 dpf and 7 dpf combined) (p=0.007,

t56 =2.8). Female hemolysis and male hemolysis were not significantly different at

1 dpf (p=0.10, t27 =1.7), but hemolysis was significantly higher in females at 7 dpf

(p=0.031, t27 =2.3) (Fig. 3).

7.0

6.5

6.0

Hemoagglutination Titer 5.5

5.0 Digestive Nondigestive

Figure 2. Hemoagglutination titers among males and female corn snakes at 1 dpf (digestive) and 7 dpf (nondigestive). Males are shown in the filled symbols. Females are shown in the open symbols. Data are plotted as mean ± SE. Hemoagglutination was higher 1 dpf for combined female and male samples.

6.0

5.5

5.0

Hemolysis Titer Hemolysis 4.5

4.0 Digestive Nondigestive

Figure 3. Hemolysis titers among females and male corn snakes at 1 dpf (digestive) and 7 dpf (nondigestive). Males are shown in the filled symbols. Females are shown in the open symbols. Data are plotted as mean ± SE. Female hemolysis was significantly higher overall and at 7 dpf.

Discussion

By examining the hemoagglutination and hemolysis titers at both digestive and nondigestive states, this study provides new insight into the effects of digestion on innate immunity. I demonstrate that hemoagglutination is significantly higher during the digestive state (Fig. 2). This is in support of our first hypothesis which posited that an organism’s innate immune system is upregulated during digestion. Because food is covered with potentially pathogenic microbes, this allocation toward immune function may be an adaptive response to digestion

(Conway, 1997; Barboza et al., 2010).

Energy allocation to innate immunity may be a component of the integrated response to digestion. Many organs, such as those of the gut, pulmonary, and cardiovascular systems, are required for digestion, and all undergo dramatic 7 shifts in size and function due to digestion (Secor, 2009). Enzymatic activity increases greatly as well as oxygen consumption which serves as a proxy for

SDA. Although the energy characterized as SDA has been shown to be devoted to these many processes, some of it may be devoted to upregulating the immune system.

My results add to the growing literature on the interplay between food and immunity. Diet has been shown to affect immune function in various ways across taxa. In insects, restriction of calories and starvation has been shown to decrease immune function (Chambers & Schneider, 2012; Aryes & Schneider,

2009). Furthermore, diet composition can affect immune function—increased protein intake leads to higher melanization and lysozyme (antimicrobial enzyme that attacks bacterial cell walls) levels whereas phenoloxidase (enzyme that melanizes capsules and produces parasite-killing toxic intermediates) activity was most influenced by levels of carbohydrate in the diet (Chambers &

Schneider, 2012; Cotter & Simpson, 2011). In addition, Siva-Jothy & Thompson

(2002) showed that decreased phenoloxidase activity ensued from short-term starvation in adult mealworm beetles. Nutritional status also affects host tolerance (when infection costs are mitigated by lowering host tissue damage and immune function activation) (Ponton et al., 2013). Tolerance to the bacterium, Salmonella typhiurium , increased in food-restricted Dropsophila and mutant flies (mutants exhibit normal reduced feeding habits) than in wild-type controls (Aryes & Schneider, 2009). Furthermore, nutritional status can also affect parasite virulence—parasites were more successful at reproducing in

8 nonsupplemented canaries, but food-supplemented canaries incurred a higher infection cost, exhibiting lower hematocrit (packed cell volume) (Cornet et al.,

2014).

Differences in hemoagglutination and hemolysis between males and females

(Fig. 3) may be explained by higher levels of testosterone in males. Testosterone has been shown to reduce the immune response in other vertebrates—males can exhibit reduced immune function, such as antibody production, cytokine production, and inflammatory responses (Shuurs & Verheul, 1990; Folstad &

Karter, 1992). Testosterone has also been shown to be higher in males than in females in neonatal/juvenile snakes (Taylor & DeNardo, 2005). Thus, differences in testosterone among males and females may explain the differences found in hemolytic activity.

While hemoaggutination and hemolysis are both parts of innate immunity and are generally correlated (Matson et al., 2005), independent variables can affect them differently. Agglutination and lysis are generally correlated due to the linked mechanism of complement activation (resulting in lysis) by means of natural antibodies (resulting in agglutination). However, lysis and agglutination respond differently to temperature and reproductive status (Butler et al., 2013;

Stahlschmidt et al. 2013). Similarly, my results demonstrate that hemoagglutination was significantly higher at 1 dpf (Fig. 2), but hemolysis was not (Fig. 3). Complement activity and, subsequently, lysis is influenced by a variety of other immune factors than simply natural antibodies (Butler et al.,

2013). Similar to what was found in this study, Butler et al. (2013) found

9 instances of decoupling where agglutination response was high but lysis was

low.

In sum, my results indicate that some energy from the SDA response is devoted

to the upregulation of the energetically costly immune system, potentially to

combat increased foreign microbe exposure. Furthermore, sex-based variation

may be present in the levels of innate immune function response due to

differences in circulating androgens among males and females. I advocate future

research into the mechanisms underlying the devotion of energy from the SDA

response to innate immunity via cell-mediated (Martin et al., 2003) and humoral

responses, as well as research examining immune-related shifts due to digestion

during the breeding season when testosterone is higher. Lastly, I advocate

research examining the effects of digestion on adaptive immunity as well as other

aspects of innate immunity, such as bacterial-killing assays, to provide a more

complete picture of the interactions between the digestive and immune systems

(Forsman et al., 2008).

Acknowledgements

I would like to thank the Department of Biology at Georgia Southern University

(GSU) for funding via the Chandler Scholarship. I also thank the Office of

Research Services and Sponsored Programs at GSU for start-up support for

ZRS and Lindsey Holcomb for animal husbandry. I am also very appreciative to

Tony Mills at the Low Country Institute for the loan of animals.

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