influence of an enteric infection on the intestinal...

Influence of an enteric infection on the intestinal epithelial

barrier function of severely malnourished mice

Thesis by

Emma Onverwagt

S2411865

In partial fulfilment of the requirements for the degree of

MSc Geneeskunde, Rijksuniversiteit Groningen

Supervisor: R.H.J. Bandsma, MD, PhD

Department of Physiology and Experimental Medicine

The Hospital for Sick Children, Toronto, Canada

April 2017

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ABSTRACT

Severe acute malnutrition (SAM) is a global problem that is associated with a high mortality

rate in children. Even after hospitalization, up to 35% of SAM patients do not survive.

Mortality is linked to the development of diarrhea indicating intestinal dysfunction. Intestinal

pathogens are frequently found but the pathophysiology of the intestinal dysfunction during

enteric infection in the context of SAM is not well understood. In this study, several pilot

experiments were performed to develop a murine model of SAM and enteric infection. It

focused on the effects of this double insult on intestinal histology and intestinal epithelial

barrier function. A model was developed in which mice received a diet containing 1% protein

and were given a seven-day infection with 10⁷ CFU C. rodentium. The combined results of

the pilot projects revealed that enteric infection caused a higher infection load in

malnourished mice compared to control mice associated with more diarrhea, epithelial cell

damage and an attenuated distribution of claudin 3, a marker of epithelial barrier function. It

had no influence on the distribution of claudin 7, another marker of epithelial barrier function,

and the amount of claudin 3 and claudin 7. Furthermore, enteric infection caused weight loss,

crypt hyperplasia and goblet cell loss. In future, this murine model could be used to test novel

interventions against SAM and hence lower its case fatality rate.

SAMENVATTING

Ernstige acute ondervoeding is een globaal probleem dat wordt geassocieerd met een hoge

kindersterfte. Zelfs na ziekenhuisopname overleeft 35% van de ernstig ondervoede kinderen

het niet. Sterfte is gerelateerd aan het ontstaan van diarree wat duidt op darmdisfunctie. Er

worden vaak darmbacteriën gevonden, maar de precieze pathofysiologie van de

darmdisfunctie tijdens gastro-intestinale infectie en ernstige ondervoeding is onbekend. In dit

onderzoek zijn verschillende proefexperimenten verricht om een diermodel van ernstige

ondervoeding en gastro-intestinale infectie te ontwikkelen. Ook de invloed van deze dubbele

belasting op de morfometrie en epitheliale barrière van de darm werd onderzocht. Er werd een

diermodel ontwikkeld waarin muizen een dieet met 1% eiwit kregen en een 7-daagse infectie

met 10⁷ CFU C. rodentium. De gecombineerde resultaten van de proefexperimenten lieten

zien dat gastro-intestinale infectie een hogere infectiebelasting veroorzaakte in ondervoede

muizen dan in controle muizen. Dit werd geassocieerd met meer diarree, epitheliale cel

beschadiging en een veranderde verdeling van claudin 3, een marker van darmdisfunctie.

Verder resulteerde gastro-intestinale infectie in gewichtsverlies, crypt hyperplasie en

slijmbekercel verlies. Het had geen invloed op de verdeling van claudin 7, een andere marker

van darmdisdisfunctie, en de hoeveelheid claudin 3 en claudin 7. In de toekomst kan dit

diermodel verder worden ontwikkeld en gebruikt worden om nieuwe interventies tegen

ernstige ondervoeding te testen en hierdoor de lethaliteit te verlagen.

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TABLE OF CONTENTS

Introduction p. 4-8

Materials and methods p. 9-11

Results p. 12-19

Discussion p. 20-24

Conclusion p. 25

Acknowledgements p. 26

Bibliography p. 27-30

Appendix I p. 31

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INTRODUCTION

Severe acute malnutrition and enteric infection:

Severe acute malnutrition (SAM) is a global problem with a high mortality rate. Its under-five

mortality rate is estimated to be more than half a million child deaths per year (1), an amount

that is even expected to increase in future (2). SAM can present itself as two distinct

phenotypes or as a combination of both. It can present itself as marasmus, which is defined by

a weight-for-height score of three standard deviations below the median or as kwashiorkor

what is characterized by bilateral nutritional edema (3). Despite following WHO guidelines,

the inpatient mortality of SAM remains as high as up to 35% (4), showing that current

guidelines are often insufficient. Even though standardized WHO guidelines have greatly

improved child mortality in SAM, most guidelines are solely based on expert opinion and the

scientific evidence is weak (4,5). Further research on SAM and its underlying

pathophysiological mechanisms is essential to improve treatment and thereby lower inpatient

mortality.

There is growing recognition that enteric infection is involved in the pathogenesis and high

inpatient mortality of SAM. SAM’s etiology is complex and is not only caused by an overall

diminished energy intake or a protein-deficient diet, but likely also involves exposure to

microbes and subsequent repeated enteric infections (6). Many children in low- and middle-

income countries have poor sanitation and hygiene what can result in an incompletely defined

syndrome called environmental enteric dysfunction (EED) (8). This poorly understood

disorder is characterized by intestinal inflammation, reduced absorptive capacity and reduced

intestinal epithelial barrier function (10). It can be accompanied with weight loss or altered

stool consistency, but patients often do not have clear signs of illness (6). EED is likely

caused by continuous exposure to entero-pathogens (11) and is thought to be a reason why

current therapeutic interventions in severely malnourished children often fail (11).

Malnutrition and enteric infection can form a vicious circle eventually leading to severe

malnutrition and death (5,8,9; figure 1). Malnutrition weakens the immune system which

causes greater susceptibility to infections and infections cause anorexia, reduced nutrient

absorption and nutrient loss which in turn worsens malnutrition (7). Research shows that

death amongst children with SAM is mostly a result of an infection on top of SAM with an

enteric infection being the most prominent one (7,9).

Figure 1. The vicious cycle of malnutrition and enteric infection, obtained from (12)

http://www.communityledtotalsanitation.org/resource/environmental-enteric-dysfunction-overview

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In addition to increasing knowledge on the role of enteric infection, there is also increasing

knowledge on the intestinal microbiome during malnutrition. The intestinal microbiome refers

to the genetic material of all commensal, symbiotic and pathogenic microorganisms that can

be found in one’s gut. Several studies have shown that the microbiome of malnourished

children differs from non-malnourished children because it is more immature (13,14). This

underscores the influence of microbes and enteric pathogens on the development and clinical

presentation of SAM.

The intestinal epithelial barrier:

The intestinal epithelial barrier forms the luminal surface of the intestine and consists of a

single layer of intestinal epithelial cells (IECs) shielded from the lumen by a thick layer of

mucus (15). Its two main functions are protection against harmful substances and facilitation

of transport of water, nutrients and ions. Apart from IECs, the intestinal epithelium also

consists of several specialized cells such as goblet cells that produce mucus and thereby form

a protective mucus layer (16).

The IECs of the intestinal epithelium are connected by tight junctions to keep out endotoxins,

inflammatory cells and other unwanted substances (15). Tight junctions (TJs) connect IECs at

the apical side (17) and consist amongst others of claudin proteins, that are considered to be

the backbone of TJs (13, figure 2). These transmembrane proteins are anchored to the cellular

skeleton by Zonulin-1, a scaffolding tight junction protein (15). Besides TJs, IECs are also

connected by adherens junctions and desmosomes that provide additional strength to keep the

cells together (18).

Figure 2. Overview of cell junctions, adapted from (19).

There are three different mechanisms that cause dysfunction of the intestinal epithelial barrier

during disease (figure 3). Two of these are dependent on TJs: the leak pathway and the pore

pathway (18). The leak pathway consists of TJs that seal the paracellular space and the pore

pathway consist of TJs that form pores and thereby regulate paracellular transport.

Furthermore, there is also a mechanisms that is independent of TJs and is characterized by

epithelial damage (18). A decrease in TJs of the leak pathway or a disruption of TJs increases

paracellular transport and thereby compromises the intestinal epithelial barrier (20). The pore

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pathway is more complex as both an increased and decreased amount of TJs can increase

paracellular transport by respectively pore formation and the formation of gaps (18). During

tight junction disruption, there are relatively more TJs in the cytosol and less on the epithelial

membrane because the transport of TJs to the cell membrane is distorted (21). An increase in

paracellular transport and hence intestinal permeability has been found to be a major

contributor to diarrhea and malnutrition (17).

Figure 3. Possible mechanisms of epithelial barrier dysfunction during disease

Severe acute malnutrition, enteric infection and the intestinal epithelial barrier:

Previous, unpublished research conducted by dr. Bandsma’s lab examined the changes in

epithelial barrier function in mice with severe acute malnutrition. Compared to control mice,

malnourished mice suffered weight loss, decreased length, villous blunting, crypt hypoplasia

and colonic goblet cell loss. Malnourished mice also had an increased permeability of the

epithelial barrier and a decreased expression of the tight junction proteins claudin 3 and

claudin 7 in the colon. Similar morphometry (10,22–26) as well as an increase in intestinal

permeability (24,27–29) has been found previously in children with severe acute malnutrition.

A study by Demehri et al also found a decrease in TJ expression in an animal model of

malnutrition per se (30).

The pathophysiology of intestinal epithelial barrier dysfunction during the double insult of

enteric infection and SAM is not well understood. Chronic enteric infection, such as

environmental enteric dysfunction (EED), has been characterized by small intestinal villous

atrophy, crypt hyperplasia and lymphocytic infiltration of the small intestinal lamina propria

(7,8,11,31). Moreover, there is data to suggest that enteric infection alone or in combination

with malnutrition contributes to an increase in intestinal permeability in children (24,27–29).

Lastly, a study by Brown et al. (32) found an increased or decreased expression of several

tight junctions during moderate acute malnutrition (MAM) in combination with an enteric

infection.

Animal model: To further examine the effects of enteric infection on intestinal dysfunction during severe

acute malnutrition, an animal model can be used. An animal model can also potentially be

used for future mechanistic studies testing novel interventions against SAM. In the 1960s and

1970s, human studies have been used to study intestinal dysfunction during malnutrition and

EED (24,26), but they are currently deemed unethical because of the need of invasive

procedures such as intestinal biopsies. To date, there has been a scarcity of studies making use

of animal models to study the interaction between malnutrition, intestinal infections and

intestinal function. In a previously mentioned animal model of Brown et al. (32) an enteric

infection with Bacteroidales species and E. coli was studied (32). Another pathogen that is

considered a golden standard to induce enteric infection in mice is Citrobacter rodentium. C.

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rodentium is a gram negative bacterial pathogen closely related to the human pathogens

enteropathogenic E. Coli and enterohemorrhagic E. Coli (33). It first attaches to the caecum

and later spreads towards the distal colon (34). It causes epithelial cell death and an increase

in proliferation and loss of differentiation resulting in crypt hyperplasia and goblet cell loss

(35, figure 4). Furthermore, it causes weight loss (36,37) as well as a disruption of tight

junction proteins (21). Tight junction disruption has also been seen during EHEC and EPEC

infections (38–40).

Figure 4. The colonic intestinal epithelial barrier in healthy mice and mice with a C. rodentium infection,

adapted from (41).

Aims:

This study aims to develop an animal model of severe acute malnutrition (SAM) and enteric

infection. Such model will enable greater understanding of the pathophysiology of intestinal

dysfunction during enteric infection in the context of SAM. Consequently, mechanistic

studies could be conducted to find novel interventions against SAM which will hopefully lead

to human trials and eventually to lower mortality. This study also aims to characterize the

intestinal dysfunction during the dual insult of enteric infection and SAM. As animal model a

previously developed murine model of SAM will be used and an enteric infection with C.

rodentium will be added.

My research questions are as follows:

1) What are a suitable infectious dose and infection length to induce an enteric infection

with C. rodentium in severely malnourished mice?

A dose and length are considered suitable if

o C. rodentium can be detected in stool at the end of the experiment

o clinical symptoms such as weight loss or diarrhea can be seen

o the infection is not lethal during the experiment

2) What is the influence of enteric infection on intestinal morphometry and the intestinal

epithelial barrier function and are malnourished mice more affected than control mice?

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I hypothesize that a suitable infectious dose will be 10⁸ CFU and a suitable infection length 10

days. These correspond to respectively the minimum infectious dose mentioned in previous

studies (42) and the height of infection. Furthermore, I expect that the enteric infection will

cause villous blunting, crypt hyperplasia, goblet cell loss and epithelial cell damage and that it

will lower tight junction protein expression and attenuate tight junction distribution. Finally, I

postulate that changes will be greater in malnourished than control mice.

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MATERIALS AND METHODS

Animal model:

Male C57BL/6 mice were weaned after 21 days and put on either a protein deficient or

control diet. The protein deficient diet contained 1% protein and the isocaloric control diet

contained 18% protein. An overview of the nutritional composition of the mice’ diets can be

found in Appendix I. During the experiment mice had free access to water and their assigned

diet and their weight was determined regularly. Four days after weaning, the mice were either

infected with C. rodentium or not infected and eight and eleven days after weaning stool was

collected. 11 or 14 days after weaning, the mice were sacrificed through carbon dioxide

inhalation and their tissue and feces were harvested for further analysis. All mice were held in

an environment with a light: dark cycle of 14: 10, humidity between 50 and 60 % and a

temperature between 23 and 25 °C. The animal handling was done according to the Canadian

Council on Animal Care guidelines and approval was given by the Animal Care Committee of

SickKids.

C. rodentium infection:

C. rodentium from a frozen stock obtained from Dr. Kim’s lab (SickKids, Canada) was plated

on Luria-Bertani (LB) agar to obtain single colonies. A single colony was then inoculated into

10 ml of LB broth and grown statically for 16 hours at 37 °C. Afterwards, the concentration

of C. rodentium was measured using a Nanodrop 2000c spectrophotometer and adapted until

the OD₆₀₀ was 0.7 ± 0.02 nm corresponding to a C. rodentium concentration of 10¹⁰ CFU/ 100

μl. The solution of C. rodentium was diluted 100 or 1000 times in phosphate-buffered saline

(PBS) to obtain concentrations of respectively 10⁸ CFU/ 100 μl and 10⁷ CFU/ 100 μl. In

previous studies, mostly a C. rodentium concentration between 10⁸ and 10⁹ CFU was used to

induce an enteric infection (36,37,43,44). Since the mice in our study were younger than the

mice used in aforementioned studies, as start dose 10⁸ CFU C. rodentium has been chosen.

Furthermore, it has been reported that the infection plateaus at day 7 and starts to clear 3 days

later (34) and that colonic hyperplasia can be seen between 6 and 20 days post-infection (45).

Due to these characteristic as a start an infection duration of 10 days was chosen. During the

fourth day post-weaning the mice that were assigned to be infected with C. rodentium were

orogastrically infected with 100 μl / 10 gram of the C. rodentium solution of 10⁸ CFU/ 100 μl

or 10⁷ CFU/ 100 μl. The other mice were orogastrically inoculated with 100 μl / 10 gram of

sterile PBS. After inoculation, the mice were monitored daily before sacrifice on day 11 or 14.

C. rodentium colonization:

Of the mice that were infected with C. rodentium, fecal pellets were collected on day 4, day 7

and during sacrifice (for the mice of pilot project 1 and 2) and on day 4 and during sacrifice

(for the mice of pilot project 3). Of the mice that were not infected by C. rodentium, fecal

pellets were collected during sacrifice. All fecal pellets were stored at -80 °C until further

analysis. The relative amount of C. rodentium in the fecal pellets was determined by a real

time qPCR using an espB primer, which measures a virulence protein that is specific for C.

rodentium (46).

Firstly, bacterial RNA was extracted using a Direct-zol RNA miniprep plus kit (Zymo

research). The concentration and purity (A260/A280 ratio) of isolated RNA were measured

using a Nanodrop 2000c spectrophotometer. Hereafter, cDNA was created by using qScript

supermix (Quanta Biosciences, MA, USA). A RT qPCR was performed using a CFX96

C1000 Thermal Cycler (BioRad). C. rodentium was quantified relative to total bacteria using

an universal eubacteria primer (47). This quantification method has been described in a recent

10

paper by Sherman et al (48). For the rest of the analyses, only mice that were infected with C.

rodentium according to above mentioned analysis were included.

Histology:

Histology was done to examine villous height in the jejunum and crypt depth and goblet cell

density in the colon. After tissue harvesting, the proximal end of the jejunum and the distal

end of the colon were cut off and fixed in formalin overnight at 4 °C. Afterwards, tissue was

dehydrated, embedded in paraffin and sectioned at 5 µm. The sections were deparaffinized

and hydrated through xylene and graded alcohols. The jejunum sections were stained with

Hematoxylin and Eosin staining and the colon sections with Periodic Acid Schiff staining

(Skytek, UT, USA). Stained tissue was visualized and scanned under a light microscope.

Scans were examined by using Pannoramic Viewer 1.15.4. Villous height was measured from

the top of the villous to the start of the crypt; 20 measurements per section were done. Crypt

depth was measured from the top of the crypt to the submucosal layer; 30 measurements per

section were done. Goblet cells were counted per crypt for the colon; 30 measurements per

section were done. Goblet cell counts were expressed as the mean number of goblet cells per

50 µm of crypt.

Immunofluorescence:

Immunofluorescence was done to visualize the distribution and amount of several tight

junction proteins in the jejunum and colon. Deparaffinized and hydrated colon tissue sections

were obtained as described above. Antigens were retrieved with a sodium citrate buffer. As

primary antibodies claudin 3 (Abcam, MA, USA) and claudin 7 (Abcam, MA, USA) were

used in dilutions of respectively 1:40 and 1:200. They were incubated overnight at 4 °C.

Afterwards, a fluorescently labelled secondary antibody (in a dilution of 1: 10,000) and DAPI

(nucleus staining) were incubated for one hour. The slides were visualized using an Eclipse

Ni-U Upright microscope (Nikon).

Western blotting:

Western blotting was done to quantify the relative concentrations of several tight junction

proteins in the colon. After tissue harvesting, the medial section of the colon was cut off,

rinsed with ice cold PBS and stored at -80 °C until further analysis. The mucosal layer of the

colon was scraped off from the muscle layer with the blunt side of a razor blade. Tissue

extraction buffer (Invitrogen, CA, USA) mixed with Protease Inhibitor Single-USE cocktail

(Sigma, MO, USA) was added in an amount of 200 μl per 20 μg tissue. Proteins were isolated

from the mucosal layer by sonicating each sample three times 10 seconds. The samples were

spun down, supernatant was transferred and the protein concentrations in the supernatant were

determined by a Bicinchoninic Acid (BCA) Protein Assay (Thermo Scientific, IL, USA).

After this, the samples were diluted to a protein concentration of 20 mg/ml and proteins were

denatured by heating them up to 95 °C for 5 minutes. Protein samples were loaded on a

NuPAGE 4% - 12% Bis Tris gel and separated by electrophoresis. Afterwards, protein was

transferred to a PVDF membrane. The membrane was first blocked with milk (consisting of

dry milk and 1xTBST) and then incubated with the following primary antibodies: β-actin

(Sigma, MI, USA), claudin 3 (Abcam, MA, USA) and claudin 7 (Abcam, MA, USA). The

dilutions of aforementioned antibodies were all 1:2000. Afterwards secondary antibodies were

added in a dilution of 1:10,000. The membrane was made immunodetectable by using an ECL

Plus Kit (Invitrogen, CA, USA). Protein expression levels were detected by an Odyssey

scanner (LI-COR Biosciences, LI, USA) and quantified by using Image Studio 5.0.

11

Statistical analysis:

Statistical analyses were performed using Excel 2016 and GraphPad Prism Software 6.00.

Unless stated otherwise, all results are expressed as the mean ± standard error of the means

(SEM). Comparisons between two groups were made using unpaired two-tailed Student’s t-

tests and comparisons between more than two groups were made by using a two-way analysis

of variance (ANOVA). P values <0.05 were considered statistically significant. In all figures,

one asterisk corresponds to a p value <0.05, two asterisks correspond to a p value <0.01 and

three asterisks correspond to a p value <0.001.

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RESULTS

Pilot projects 1 and 2

First pilot project 1 was performed to determine if a concentration of C. rodentium of 10⁸

CFU was adequate to induce enteric infection (figure 5). This dose was recommended as

minimum dose in previous studies (42). For this pilot project four malnourished mice,

receiving a diet containing 1% protein, were inoculated with 10⁸ CFU of C. rodentium. All

mice reached a humane endpoint before the end of the experiment. They lost more than 20%

of their initial weight (figure 6).

Consequently, for pilot project 2 the given dose of C. rodentium was lowered to 10⁷ CFU.

Four malnourished and three control mice were inoculated with 10⁷ CFU of C. rodentium.

During this pilot project, also a substantial amount, three out of four malnourished mice,

reached a humane endpoint before the end of the experiment. The two mice that reached a

humane endpoint on day 12 and day 13 were still included in the analyses because we only

found out during sacrifice that they did. In total the data of three infected malnourished mice

were used for the rest of the analyses.

Figure 5. Pilot projects 1 and 2

Figure 6. Survival of malnourished mice that were infected with doses of respectively 10⁸ and 10⁷ CFU of C.

rodentium. Survival is defined as the number of mice that did not reach a humane endpoint. n=4 for both graphs

All mice that were inoculated with 10⁷ CFU C. rodentium had detectable bacteria in their

stool. After inoculation with the same dose of C. rodentium, malnourished mice, who were

given a 1% protein diet, had a higher fecal infection load than control mice, who were given

an 18% protein diet (figure 7). In both malnourished and control mice, the infection load

increased up to day 7 post-infection after which it further increased in malnourished mice and

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slightly decreased in control mice. To make sure that the enteric infection was in the same

phase in malnourished and control mice and because too many animals reached a humane

endpoint during pilot project 2, for pilot project 3 we choose to shorten the infection to seven

days and to shorten the overall duration of the experiment to eleven days.

Figure 7. Infection load of C. rodentium in feces. Relative expressions have been calculated with qPCR analysis

and have been normalized to the total amount of bacteria with a universal eubacteria primer. n=3 for all groups.

The effect of C. rodentium infection on weight loss and diarrhea in malnourished and

control mice. Over the course of 14 days all malnourished mice lost weight and all control mice gained

weight. In figure 8A and 8B, weight loss is represented as weight loss post-infection or in

other words weight loss between day 4 and day 14 of the experiment. Malnourished mice that

were infected with C. rodentium lost most weight. They lost on average 24% of their weight

after they were infected with C. rodentium. Malnourished mice that were not infected lost less

weight during this period, on average 11%, a statistically significant difference (p<0.001).

Control mice that were infected with C. rodentium gained on average 16% of their weight

after they were infected. Control mice that were not infected gained more weight, on average

43%. This difference in weight loss was also significant (p<0.01). When comparing

malnourished and control mice, control mice lost relatively more weight after a C. rodentium

infection than malnourished mice, 19% compared to 14%. However, the euthanized mouse is

not included and therefore the weight loss of 14% of malnourished mice is likely an

underestimation.

Apart from weight loss, also stool consistency was studied. During the experiment, feces were

sampled at several time points: on day 4, 7 and during sacrifice. Malnourished mice proved to

be more prone to diarrhea than control mice. All infected, malnourished mice had diarrhea at

the end of the experiment compared to 33% of infected, control mice (figure 8C).

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Figure 8. Weight change and change in stool consistency after an enteric infection. (A) weight change, presented

as the ratio of weight change post-infection. Error bars represent the SEM, n= 25 (18%), n=3 (18% + C. rod)

n=30 (1%), n=3 (1% + C. rod). (B) ratio of weight change on day 10 post-infection. Significance has been

calculated with a student’s t-test whereby ** corresponds to p<0.01 and *** to p<0.001. (C) stool consistency,

presented as the percentage of mice with diarrhea, n=3 for both groups. Diarrhea was defined as watery, loose

stool that did not form a fecal pellet.

The effect of C. rodentium infection on crypt hyperplasia and goblet cell loss in

malnourished and control mice.

After sacrifice, colon tissue was collected, dehydrated, paraffinized, sectioned, stained and

scanned. Histological slides of infected mice were compared to slides of non-infected mice

that were obtained earlier by another investigator of Dr. Bandsma’s lab. The data of the other

student showed that malnutrition caused crypt hypoplasia (p<0.01) and goblet cell loss

(p<0.001; figure 9). Contradictory to malnutrition, the enteric infection caused crypt

hyperplasia. In control mice, the average crypt depth increased 134 μm to 194 μm (p<0.01)

and in malnourished mice from 92 μm to 147 μm (p<0.05). Furthermore, the enteric infection

caused goblet cell loss. The average amount of goblet cells decreased from 7.76 cells/50 μm

to 4.63 cells/50 μm in control mice (p<0.01) and from 4.71 cells/50 μm to 3.78 cells/50 μm in

malnourished mice (p<0.05). Lastly, in non-infected mice and in infected, control mice there

was only some epithelial damage, whereas in infected, malnourished mice, the epithelial

damage was substantial. This epithelial damage may have caused that the average crypt depth

of malnourished, infected mice has been underestimated.

15

Figure 9. Intestinal morphology after an enteric infection: crypt depth (A), goblet cell density (B) and

corresponding PAS stained colon tissue (C). Error bars represent the SEM and significance has been calculated

with a student’s t-test whereby * corresponds to a p<0.05, ** to p<0.01 and *** to p<0.001, n=6 (18%), n=3

(18% + C. rod), n=7 (1%), n=3 (1% + C. rod). The right PAS-stained colon tissue pictures were obtained by me

and the left pictures more than a year ago by another student. Due to technical problems, there was too little data

on villous height (n=1 or n=2 per group) and this is therefore left out in this thesis.

The effect of C. rodentium infection on tight junction protein expression in malnourished

and control mice. From colon tissue, proteins were extracted and the tight junction proteins claudin 3 and

claudin 7 were quantified using western blotting. Even though there is a trend that the relative

protein expression of claudin 3 and 7 is higher in the non-infected control mice, than in the

other groups this difference was non-significant (figure 10).

Figure 10. Relative expression of the tight junction proteins claudin 3 (A), claudin 7 (B) and the corresponding

western blot (C). Error bars represent the SEM and significance has been calculated using a two-way ANOVA,

n=3 for all groups.

Pilot project 3

The effect of C. rodentium infection on weight loss and diarrhea in malnourished and

control mice.

During pilot project 3, mice were fed either a protein deficient (1% protein) or control diet

(18% protein) and on the fourth day post-weaning they were either inoculated with C.

rodentium or sham-inoculated with PBS (figure 11). They were sacrificed on day 11 post-

weaning. Contrary to the previous pilot projects, none of the animals reached a humane

16

endpoint before the end of the experiment. The average infection load of C. rodentium in

feces was lower than during pilot project 2, but otherwise similar (figure 12).

Figure 11. Pilot project 3

Figure 12. Infection load of C. rodentium in feces. Relative expressions have been calculated with qPCR analysis

and have been normalized to the total amount of bacteria with a universal eubacteria primer. n=3 (1% + C. rod

and control), n=4 (18% + C. rod).

Similar to pilot project 2, all malnourished mice lost weight and all control mice gained

weight during the experiment. In figure 13, an overview of the combined results of pilot

project 2 and 3 can be found. Malnourished mice that were infected with C. rodentium lost on

average 12% of their weight after they were infected and malnourished mice that were not

infected 10% of their weight, a statistically non-significant difference (p>0.9). On the other

hand, infected, control mice gained on average 25% of their weight and control mice that

were not infected 36%, a statistically significant difference (p<0.05). Furthermore,

malnourished mice had more often diarrhea than control mice. Seven days after infection,

50% of malnourished mice had diarrhea compared to 29% of control mice.

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Figure 13. Weight change and change in stool consistency after an enteric infection. These are the combined

results of pilot project 2 and 3. (A) weight change, presented as the ratio of weight change post-infection. Error

bars represent the SEM, n= 30 (18%), n=7 (18% + C. rod) n=34 (1%), n=7 (1% + C. rod). (B) ratio of weight

change on day 10 post-infection. Significance has been calculated with a student’s t-test whereby * corresponds

to a p<0.05 and *** to p<0.001. (C) stool consistency, presented as the percentage of mice with diarrhea, n=7

(18% + C. rod), n=6 (1% + C. rod).

The effect of C. rodentium infection on crypt hyperplasia and goblet cell loss in

malnourished and control mice. After sacrifice, jejunum and colon tissue have been

collected, dehydrated, paraffinized, sectioned, stained and scanned. The jejunal villi of

malnourished mice were shorter than the villi of control mice (p<0.05, figure 14), they were

on average respectively 241 μm and 313 μm. The enteric infection did not have an influence

on villous height. Similarly to the results of pilot project 2, malnourished mice had crypt

hypoplasia (p<0.05) and goblet cell loss (p<0.05). Furthermore, the enteric infection caused

crypt hyperplasia in malnourished mice (p<0.05). The average crypt depth was 136 μm in

non-infected malnourished mice and 170 μm in infected, malnourished mice. In control mice,

the enteric infection also caused slight crypt hyperplasia. The average crypt depth went from

176 μm to 204 μm, but this was not significant. Apart from crypt hyperplasia, the enteric

infection caused slight goblet cell loss in both malnourished and control mice, but these

differences were again not significant. The average goblet cell count went from 5.06 cells/50

μm to 4.80 cells/50 μm in malnourished mice and from 3.80 cells/50 μm to 3.36 cells/50 μm

in control mice.

18

Figure 14. Intestinal morphology after an enteric infection: villous height (A) crypt depth (C), goblet cell density

(D) and corresponding H&E stained jejunum tissue (B) and PAS stained colon tissue (E). Error bars represent

the SEM and significance has been calculated with a student’s t-test whereby * corresponds to a p<0.05 and **

to p<0.01. n=5 (18%), n=4 (18% + C. rod), n=4 (1%), n=3 (1% + C. rod).

The effect of C. rodentium infection on tight junction protein expression in malnourished

and control mice. After sacrifice, colon tissue has been collected, dehydrated, paraffinized

and sectioned. Two tight junction proteins, claudin 3 and claudin 7, have been visualized

using immunofluorescence. A representative picture of the expression and distribution of

claudin 3 and claudin 7 in all groups of mice is shown below (figure 15). The color intensity

represents the amount of tight junction protein. The intensity of claudin 3, and thereby the

amount of claudin 3, seemed lower in infected, malnourished mice than in the other groups.

Besides this, there were no clear differences in amount of claudin 3 and claudin 7. In healthy

animals, most tight junction proteins were present in the cell membranes. In

immunofluorescence this can be seen as a so-called chicken wire pattern (21). Claudin 3’

distribution was normal in non-infected malnourished mice and in both groups of control

mice. Only in infected malnourished mice the distribution of claudin 3 is altered. Here, the so-

called chicken wire pattern cannot be seen, claudin 3 is distributed more evenly in the crypts

and is thus relatively more present in the cytosol and less present in the cell membrane.

Claudins 7’ distribution, on the other hand, did not appear to be influenced by malnutrition or

enteric infection.

19

Figure 15. Expression and distribution of the tight junction proteins claudin 3 (A) and claudin 7 (B) in colon

tissue, 40x magnification, n=5 (18%), n=4 (18% + C. rod, 1%), n=3 (1% + C. rod).

From colon tissue, proteins were extracted and the tight junction proteins claudin 3 and

claudin 7 have been quantified using western blotting. There were no significant differences

between malnourished and control mice as well as between infected and non-infected mice

(figure 16).

Figure 16. Relative expression of the tight junction proteins claudin 3 (A), claudin 7 (B) and the corresponding

western blot (C). Error bars represent the SEM and significance has been calculated using a two-way ANOVA,

n=5 (18%), n = 4 (18% + C. rod, 1%), n=3 (1% + C. rod).

20

DISCUSSION

Overview of results:

During the first two pilot projects, many mice reached a humane endpoint before the end of

the experiment. Due to this, the dose of the enteric pathogen given to the animals had to be

lowered to 10⁷ CFU, the length of the infection shortened to 7 days and the overall length of

the experiment shortened to 11 days. After C. rodentium infection, malnourished mice had a

higher infection load than control mice which was associated with more diarrhea, epithelial

cell damage and an attenuated distribution of claudin 3. C. rodentium infection furthermore

caused weight loss, crypt hyperplasia and goblet cell loss. After seven days weight loss was

significant in control mice and crypt hyperplasia in malnourished mice and after ten days

weight loss, crypt hyperplasia and goblet cell loss were all significant in both malnourished

and control mice. The enteric infection had no influence on the distribution of claudin 7 and

on the amount of claudin 3 and claudin 7.

Animal model:

The main challenge in the development of the animal model appeared to be the severity of the

proteindeficient diet that was given to the animals. Most malnourished mice lost so much

weight that they approached a humane endpoint at the end of the study, meaning that there

was little space for extra weight loss due to the enteric infection. The protein content of the

protein deficient diet could not be increased, because previous data of Dr. Bandsma’s lab

showed that during a 2% protein diet, changes in intestinal morphometry as well as liver

steatosis could not be observed.

To stay within the boundaries of the animal use protocol, the dose of the enteric pathogen

given to the animals had to be lowered to 10⁷ CFU, a dose lower than the dose described in

previous studies, where the dose was between 10⁸ and 10⁹ CFU (36,37,43,44). Even though,

in a previous study mice did not get infected after this dose (49), in our study mice did. An

explanation why a lower dose of C. rodentium could be used is that the mice used in this

study were only three weeks old, whereas mice used in other studies were adult mice and

likely had better defense against pathogens.

Apart from lowering the infection, the length of the infection had to be shortened from 10 to 7

days. Initially was chosen for a 10-day infection, because the height of infection is between 7

and 10 days (34) and because the effects of the infection on the intestine, such as crypt

hyperplasia, start after 6 days and increase up to 14 days (45). 10 days thus represents the

height of infection and by this time effects on the intestine must be clearly visible. After

analyzing the infection load in malnourished mice and control mice, I found that up to 7 days

the infection load increased in both malnourished and control mice, but that after 7 days this

changed and the infection load kept increasing in malnourished mice, but started to decrease

in control mice. Since I was comparing the effects of the enteric infection on malnourished

and control mice, I concluded it would be optimal if the infection was in the same phase in

both groups of mice. At ten days this is doubtful, therefore it was decided to shorten the

infection. Since most animals reached a humane endpoint around day 11 of the experiment,

the infection was shortened to 7 days and the overall experiment to 11 days.

Infection load:

The infection load was higher in malnourished mice than in control mice. This means that,

when compared to the total amount of bacteria, there are relatively more C. rodentium

bacteria in the stool of malnourished mice than in the stool of control mice and hence that C.

rodentium multiplies itself faster in malnourished than control mice. One explanation could be

that the immune system of malnourished mice is impaired (50) and hence C. rodentium is less

21

restrained to reproduce itself. Another explanation could be that the microbiota of

malnourished mice differs from the microbiota of control mice (34,51) and that the microbiota

of malnourished mice is less able to compete with pathogens (52).

The infection load of malnourished mice was approximately three times higher during pilot

project 2 than during pilot project 3. Just as the longer infection duration, this could have

contributed to the results being significant during pilot project 2 and not during pilot project 3.

It is hard to measure the exact concentration of C. rodentium and it likely varies a little bit

every time a C. rodentium dilution is made. To reduce the influence of this variation on our

results, in both pilot project 2 and pilot project 3 we inoculated all malnourished and control

mice at the same time to make sure the administered dose was equal among both groups.

Clinical symptoms and intestinal morphology: The difference in infection load between malnourished and control mice did not translate to a

similar difference in weight loss. In both groups, the enteric infection with C. rodentium

caused weight loss. This has also been seen before in previous studies about C. rodentium

(36,37). It is difficult to explain why there was significant weight loss in control mice after 7

days, but not in malnourished mice. One explanation might be that there are not many animals

included and that it is mostly variation in weight change between animals instead of an actual

difference. Furthermore, one infected, malnourished mouse has been euthanized and excluded

because of too much weight loss. The weight loss in the infected malnourished group is thus

underestimated. Lastly, malnutrition weakens the immune system resulting in less cytokine

release (53,54). This could diminish systemic effects such as weight loss (55), but this is not

sufficiently supported in literature. Even though the difference in infection load was not

associated with a similar difference in weight loss, it was associated with a difference in stool

consistency between malnourished and control mice. Malnourished mice were more prone to

diarrhea than control mice.

Apart from measuring clinical symptoms, we also characterized the influence of the enteric

infection on intestinal morphology. The enteric infection did not influence villous height.

Villous blunting is a typical symptom of environmental enteric dysfunction (EED), a

syndrome of chronic intestinal inflammation caused by repeated enteric infections (8,11). Our

enteric infection therefore did not resemble EED. As far as I could find, no previous research

has been done on the influence of a C. rodentium infection on villous height. Furthermore, the

enteric infection caused crypt hyperplasia and goblet cell loss after 10 days of infection. After

7 days only crypt hyperplasia was significant and only in malnourished mice. However, these

were pilot experiments and not adequately powered to detect significant differences. Several

other studies also demonstrated crypt hyperplasia and goblet cell loss as a characteristic of

enteric infection (34,35,41). It seems that the enteric infection influences the morphometry of

the colon, where the infection takes place, but does not seem to have an influence on the small

intestine as can be seen during chronic enteric infection such as EED.

Epithelial barrier dysfunction:

This study examined the influence of enteric infection on epithelial cell damage and the

distribution and amount of two tight junction proteins, claudin 3 and claudin 7. Both epithelial

cell damage and an altered distribution or decreased amount of these tight junction proteins

contribute to increased paracellular flux and hence epithelial barrier dysfunction (15,56). The

difference in infection load between malnourished and control mice, as discussed earlier, was

also associated with a difference in epithelial cell damage between infected malnourished and

the other groups of mice. The enteric infection caused substantial epithelial cell damage in

22

malnourished mice, that was not seen in control mice. This epithelial cell damage is

associated with an increased paracellular flux and decreased epithelial barrier (18).

The difference in infection load was also associated with a difference in claudin 3 distribution.

In infected malnourished mice claudin 3 showed a relatively lower expression on the cell

membrane than in the other groups. The normal chicken wire pattern that is usually seen

during immunofluorescence of tight junction proteins (21) disappeared. Proteins were more

evenly distributed. In previous studies a similar alteration in claudin 3 distribution after an

enteric infection has been found (57). This alteration in distribution suggests that there is

fewer claudin 3 transported to the cell membrane where it exerts its function by sealing the

paracellular space (15). Fewer claudin 3 on the cell membrane would likely result in increased

paracellular flux and hence decreased barrier function. Contrary to claudin 3, the distribution

of claudin 7 was not altered by the enteric infection. To my knowledge, the distribution of this

tight junction protein has not been studied before in relation to enteric infection.

In this study, no significant difference was found in the relative protein expression of claudin

3 and claudin 7. This contradicts with earlier obtained unpublished data of Bandsma’s lab. In

those data, a significant decreased protein expression of claudin 3 and claudin 7 was found in

malnourished mice when compared to control mice. Our pilot projects were carried out in

small numbers of animals and might therefore have been underpowered to detect significant

differences. Another possible explanation could be an inadequate loading control during

western blotting. Contrary to pilot project 2, during pilot project 3, there was no trend of

decreased tight junction expression in malnourished mice compared to control mice. A

possible explanation could be that the amount of tight junctions only start to decrease after the

mice have been malnourished for some time. During pilot project 2, mice were given a

malnourished diet for 14 days, while during pilot project 3 this was 11 days. It could be that

11 days is too short to see an effect on the amount of tight junction proteins. A possible

explanation why there are less tight junctions during malnutrition is because there is a general

lack protein and thus not enough proteins for all tight junctions to form.

There are several other studies on tight junction proteins during malnutrition and enteric

infection. In a study by Brown et al (32), malnourished mice with an enteric infection had a

decreased expression of the sealing tight junctions zonulin 1 and claudin 4 and an increased

expression of the pore-forming TJ claudin 2. An increased expression of claudin 2 was also

found in mice with solely enteric infection in a study by Prasad et al (57) and in rats with

enteric infection in a study by Fernandez-Blanco et al (58). In this last study, also a decrease

in claudin 3 and occluding was found. In a study by Demehri et al, malnourished mice had a

lower expression of claudin 5 and claudin 8 (30). In other studies on enteric infection with the

enteric pathogens C. rodentium, EHEC and EPEC, no decreased amount of tight junction

proteins was found (21,38–40).

Limitations:

The experiments in this study were pilot projects that were not adequately powered to detect

differences in quantitative outcomes. Due to this, in some analyses only a trend but no

significance could be seen. Another limitation of this study is that the epithelial barrier

function has only been characterized by epithelial cell damage and the expression and

distribution of claudin 3 and claudin 7. A more direct measurement of epithelial barrier

function, such as epithelial permeability is lacking. Epithelial permeability can be measured

with a FITC Dextran Assay. Hereby fluorescent sugar is orogastrically administered and its

concentration in blood is measured 1.5 hours later. Other, still unpublished data of dr.

23

Bandsma’s lab showed that epithelial permeability was increased 2.5-fold in malnourished

mice when compared to control mice. It is expected that enteric infection also increases

epithelial permeability and that this effect is greatest in malnourished mice. It would have

been insightful when this hypothesis could have been tested. In these pilot projects a FITC

Dextran Assay has not been done because FITC and immunofluorescence cannot be

combined due to interfering fluorescent dyes. We chose to perform immunofluorescence as

this is a non-quantitative tool and therefore a reasonable choice for pilot experiments to get a

visual sense of the effect of the enteric infection.

There are some limitations related to the techniques used. During pilot project 2, the crypt

depths and goblet cell densities of infected mice were compared to previously obtained crypt

depths and goblet cell densities of non-infected mice. The analyses thus have been done by

different investigators which could have led to inconsistencies in the results. Also, the staining

protocol has been changed in between, which could have also led to inconsistencies. All

analyses of pilot project 3 have been done by me, so in this pilot there is no inter investigator

or staining inconsistency. When comparing the results of pilot project 2 and 3 some

inconsistencies can be seen. In pilot project 2 the goblet cell density of control mice is around

8 cells/50μm whereas in pilot project 3 this is around 5 cells/50μm. Since it is very unlikely

that the goblet cell count increases that much in three days, this is probably because of a

combination of inter-animal variation, inter-investigator inconsistency and inconsistency in

staining. The histology results of pilot project 2, especially the goblet cell density, should thus

be interpreted with caution. Apart from histology, also immunofluorescence could have been

improved. In this study, an epifluorescence microscope has been used. A confocal

microscope, however, would have created clearer images that better visualize protein

distribution.

As said earlier, an explanation for the contradiction between the western blot results in this

study and previous literature could be because of an inadequate loading control. As loading

control beta-actin has been used, a protein involved in a cell’s cytoskeleton. As can be seen in

the western blot results in this study, the protein concentrations of beta-actin are very variable,

a variability that cannot be explained by loading mistakes. Several other studies have also

found that beta-actin is variable in intestinal tissue and therefore not an adequate loading

control (59–61). It is a bit unclear what is the best substitute, in studies by Kouadjo et al and

Wang et al a few alternatives are listed (60,61). Another explanation is related to technical

irregularity. For western blot preparation, intestinal tissue must be lysed. It could be that I

lysed the tissue not long enough, so that only the tight junctions in the cytosol became free

and only part of the beta-actin which is in the cytoskeleton and cell nucleus and part of the

tight junctions in the cell membrane.

Future directions:

Several pilot experiments were carried out to develop an animal model of SAM and enteric

infection. A main challenge appeared to be to stay within the boundaries of ethical animal use

and hence the infection could not be too harsh or too long. For future purposes, I would

recommend using a C. rodentium dose of 10⁷ CFU and an infection length of 7 days. After

this dose and length, the enteric pathogen could be detected in feces, the infection was in the

same phase in malnourished and control mice, changes in intestinal morphology and epithelial

barrier function could be seen and the infection was not lethal. Not all changes in morphology

and barrier function were significant after this dose and length, but these were pilot projects

that were not adequately powered to detect quantitative differences.

24

During pilot project 3, we decided to shorten the overall duration of the experiment from 14 to

11 days. We did this because we were afraid that when we would adhere to a 14-day

experiment we would have to euthanize too many animals. During pilot project 3, mice

generally lost less weight than during pilot project 2. Based on the combined results of pilot

project 2 and 3, it seems that shortening the overall duration of the experiment from 14 to 11

days is not an absolute necessity. Since all data of non-infected mice for a 14-day experiment

have already been collected, including data on intestinal permeability, it might be better to

adhere to the 14-day experiment for our future mice model.

As said before, some techniques should be improved in future. Measuring villous height,

crypt depth and goblet cell density should be done in a more consistent manner. Furthermore,

one must find out if changed tissue lysis time or a changed loading control can form a

constant control for western blotting. Measuring intestinal epithelial permeability provides

more insight in the epithelial barrier function and hence should be implemented. Also,

assessing proliferation, differentiation and cell death could improve our knowledge on the

intestinal pathophysiology during enteric infection on top of SAM. With abovementioned

recommendations, improvements and additions our mice model of severe malnutrition and

enteric infection can be improved. Thereafter, it can be used to better understand the

pathogenesis of intestinal dysfunction during the double insult of SAM and enteric infection.

By better understanding the pathogenesis, possible therapeutic interventions could be

developed. Those conceivable interventions could subsequently be tested in mechanistic

animal studies and when promising in human trials. Ultimately, it could improve treatment of

malnourished children with enteric dysfunction and hence lower child mortality.

25

CONCLUSION

The first aim of this study was to develop a murine model to examine the effects of an enteric

infection on the intestinal dysfunction during SAM. As starting point an already existing

murine model of SAM was used and as enteric pathogen C. rodentium was chosen. After a

few pilot projects, a dose of 10⁷ CFU and an infection length of 7 days were considered a

suitable dose and infection length. With this dose and length, C. rodentium could be detected

in stool, part of mice developed diarrhea, control mice had significant weight loss and the

infection was not lethal during the experiment. With a higher dose (10⁸ CFU) and longer

infection (10 days), too many animals reached a humane endpoint before the end of the

experiment.

The second aim of this study was to examine the effects of an enteric infection on intestinal

morphometry and the intestinal epithelial barrier function and whether malnourished mice

were more affected than control mice. The combined results of the pilot projects revealed that

the enteric infection caused crypt hyperplasia, goblet cell loss, epithelial cell damage and an

altered distribution of the tight junction claudin 3. It did not influence villous height, the

distribution of the tight junction claudin 7 and the amount of tight junctions. Malnourished

mice had a higher infection load than control mice. This was associated with more diarrhea,

epithelial cell damage and an attenuated distribution of claudin 3. In future, this model can be

used to gain more insight in the pathophysiology of children with SAM and enteric

dysfunction, to develop new interventions and to test those.

26

ACKNOWLEDGEMENTS

This thesis would not have been possible without my supervisor, dr. Robert Bandsma. I would

like to thank him for giving me the opportunity to be part of his research team at SickKids and

for providing me with valuable and constructive feedback throughout my research internship.

Furthermore, I would like to thank all my colleagues of the Bandsma lab for their guidance

and sociability during my stay. I would especially like to thank Nathan Swain and Lijun Chi

for teaching me all necessary lab techniques and for their passionate guidance and assistance

during the experiments.

27

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31

APPENDIX I

Nutritional components

(g/kg)

18% protein diet 1% protein diet

Casein 207 11.5

DL-Methionine 2.70 0.36

Sucrose 350 350

Corn Starch 251.3 426.54

Maltodextrin 50 50

Corn Oil 52.60 54.2

Cellulose 41.06 60.6

Vitamin Mix, Teklad

(40060)

10 10

Ethoxyquin, antioxidant 0.01 0.01

Mineral Mix, Ca-P

deficient (79055)

13.37 13.37

Calcium Phosphate,

dibasic

17.36 22.32

Calcium Carbonate 4.6 1.1 Table 1. Nutritional composition of the mice diets

influence of an enteric infection on the intestinal...

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