Gut Physiology from a Pathogens Point of View - Dr. Adam Moeser, North Carolina State University, from the 2014 Boehringer Ingelheim North Carolina Swine Health Seminar, August 15, 2014, Wrightsville Beach, North Carolina, USA. More presentations at http://www.swinecast.com/2014-boehringer-ingelheim-carolina-swine-health-seminar

1. Adam Moeser DVM PhD
Associate Professor of
Gastrointestinal Biology & Swine Medicine
Department of Population Health & Pathobiology

2. Pathogenesis of Disease: Interactions Between
Pathogens, Host, and Environment
Pathogen
Environment
Susceptible
Hosts

3. Focus Points
• Mechanisms of Infectious
Enteric Disease
– Normal physiology
– Pathophysiology of diarrheal
disease
• The impact and biological
mechanisms by which
stress predisoposes
animals to disease:
– Epithelial barrier function
– Immune function
– Early life stress and long-term
development of pig GI
system
– Enteric disease susceptibility
– Mechanisms
Stress

4. Intestinal Barrier Function

5. Intestinal Absorption and Secretion:
Relevance to Diarrhea
Absorption occurs
predominantly in
the villus epithelium
Secretion occurs
predominantly in
the crypt epithelium
Relevance: pathogens that
damage absorptive villus
epithelium induce
malabsorptive diarrhea:
PEDv, Coccidiosis, TGEv,
Rotavirus
glucose
Amino acids
Relevance: bacterial toxins
and inflammatory stimuli
elicit massive Cl-, HCO-,
3
and water secretion from the
crypts: e.g. E. coli, S.
Typhimurium, etc
-
HCO3

6. The Gastrointestinal Barrier
H Nutrients Pathogens Toxins 20
HCO3-
Circulation
Intrinsic Barrier Immunologic Barrier
Tight Junctions
Extrinsic Barrier
Microbiota
Immune System
Mucus Antimicrobial
peptides
IgA
Intestinal
Lumen
Epithelial
Cells
Lamina
Propria
Cl-
Enteric nervous system

7. Mechanisms of Intestinal Secretion
ETEC Rotavirus
Bacterial toxins and
Inflammatory products
Moeser AJ and Blikslager, JAVMA 231:56-67, 2007

8. Secretory Diarrhea: Enterotoxigenic E. Coli

ETEC produces enterotoxins: STa, STb, LT

Enterotoxins bind to intestinal epithelial cells
and elicit secretion of electrolytes and block
Na+ absorption resulting in massive fluid loss
into the intestinal lumen

Minor histological damage is seen in acute
disease
Cl-
H2O
HCO3
-
Pathogenesis
E. coli
STa LT STb
Na+
X

9. Mechanisms of Intestinal Absorption
ETEC
Moeser AJ and Blikslager, JAVMA 231:56-67, 2007
Rotavirus

10. Malabsorptive Diarrhea
• Viral infections (PED, TGE,
Rotavirus) commonly cause villus
blunting and malabsorption
• Viruses can produce enterotoxins
that stimulate secretion
– E.g. Rotavirus NSP4
• Coccidiosis destroys intestinal
absorptive epithelium
• Brachyspira Hyodysenteriae
(swine dysentery) causes
malabsorptive diarrhea
Villous
Atrophy
Mucosal
surface area
Nutrient and water
absorption
Diarrhea
Cl-
H2O
Sanford
Control
Coccidiosis

11. Inflammatory Diarrhea:
Salmonella Typhimurium
Progression to MALABSORPTION
Subacute/Chronic
lesions
H20
Cl-
S typhimurium
IL-8 IL1-β
INFLAMMATION
HCO3-
HYPERSECRETION
PGE2
HCO3-
H20
Cl-
Recruitment of
neutrophils

12. Salmonella Enteritis
Healthy Control Salmonella Typhimurium
enteritis

13. Pathogenesis of Disease: Interactions Between
Pathogens, Host, and Environment
Pathogen
Environment
Stress
Susceptible
Hosts

14. Stress is a Major factor in the Onset and Exacerbation of
GI Disease in Animals and People
Stress
Stress
http://www.stress-management-for-health.
com/physical-effects-of-stress.html
Poor weight gain/feed conversion
Susceptibility to GI Infections
Diarrhea
Hyper-activated or
Suppressed Immune response
Functional and/or
inflammatory GI Disorders
The biological mechanisms by which stress causes disease remain poorly understood

15. Intestinal Stress Physiology Lab
• Long-Term Goal: Understand the biological mechanisms
by which stressors impact gut health

16. Weaning is the Most Profound Stress a Pig
Encounters in Production

17. Weaning Stressors
Maternal separation
Change in
environment
Fighting and
establishment
of social hierarchy
Increased exposure
to pathogens
Abrupt transition in diet
Transportation
stress

18. Post-Weaning Health Challenges in the Pig
How does production stress impact
gut defense and disease
susceptibility?

19. The Gastrointestinal Barrier
H Nutrients Pathogens Toxins 20
HCO3-
Circulation
Intrinsic Barrier Immunologic Barrier
Tight Junctions
Extrinsic Barrier
Microbiota
Immune System
Mucus Antimicrobial
peptides
IgA
Intestinal
Lumen
Epithelial
Cells
Lamina
Propria
Cl-
Enteric nervous system

20. Compromised Barrier Under Stress:
HCO3-
Nutrients Pathogens Toxins
H20
Intestinal
Lumen Cl-
Epithelial
Cells
Lamina
Propria
Fluid
Loss
“Leaky Gut”

21. The Gastrointestinal Barrier
H Nutrients Pathogens Toxins 20
HCO3-
Circulation
Intrinsic Barrier Immunologic Barrier
Tight Junctions
Extrinsic Barrier
Microbiota
Immune System
Mucus Antimicrobial
peptides
IgA
Intestinal
Lumen
Epithelial
Cells
Lamina
Propria
Cl-
Enteric nervous system

22. Compromised Barrier Under Stress:
HCO3-
Nutrients Pathogens Toxins
H20
Intestinal
Lumen Cl-
Epithelial
Cells
Lamina
Propria
Fluid
Loss
“Leaky Gut”

23. Measurement of Intestinal Epithelial Barrier Function:
Ussing Chamber Technique
Intestinal
Tissue
TER
Ohm’s law: V= IR
mV = μA * Ω
FITC-Dextran
Supernatant
3H-Mannitol
14C-Inulin
Increased Flux = compromised gut barrier
Decreased flux = Intact gut barrier

24. Weaning Stress Breaks Down Intestinal Barrier Function
1 0 0
8 0
6 0
4 0
2 0
0
T E R ,  . c m
0 . 0 6
0 . 0 4
0 . 0 2
2
. h
Moeser et al. Am J Physiol Gastrointest Liver Physiol. 292:G173-81
2
U n w e a n e d
W e a n e d
*
0 . 0 0
3
H -m a n n i t o l f l u x ,  m o l . c m
*
U n w e a n e d
W e a n e d
Increased
Intestinal Permeability
Weaning

25. Influence of Chronic Production Stressors on Intestinal
Barrier Function in Pigs
Mixing and Crowding Stress Chronic Heat Stress
P<0.01 P<0.01
Control Mixing/crowding
Stress
Control (TN) Heat Stress

26. Influence of Chronic Production Stressors on
Intestinal Glucose Transport
Mixing and Crowding Stress Chronic Heat Stress
P<0.01 P<0.01
Control (TN) Heat Stress
ΔIsc, uA/cm2
ΔIsc, uA/cm2
Mixing/crowding
Stress
Control

27. Influence of Pig Age on
Weaning-induced Intestinal Injury

28. Delayed Weaning Ameliorates Weaning-Induced
Intestinal Barrier Dysfunction
0 . 0 5
0 . 0 4
0 . 0 3
0 . 0 2
0 . 0 1
0 . 0 0
2
. h
3
H -m a n n i t o l f l u x ,  m o l . c m
U n w e a n e d
W e a n i n g s t r e s s
*
E W S
( 1 6 d w e a n i n g )
L W C
( 2 8 d w e a n i n g )
Moeser et al. Am J Physiol Gastrointest Liver
Physiol. 293:G413-21
1 5 1 8 2 1 2 3 2 8
0 . 0 8
0 . 0 6
0 . 0 4
0 . 0 2
0 . 0 0
2
. h
3
H -m a n n i t o l f l u x ,  m o l . c m
W e a n in g A g e
Smith et al. Am J Physiol Gastrointest Liver Physiol
2010;298:G352-G363

29. Experimental Design: Long-term Impact of Weaning
Age on Intestinal Barrier Function
112
d
Late Weaned Control (LWC)
Weaned at 28d
Early Weaning Stress
(EWS)
Weaned at 16 d
Intestinal permeability measurements
1d 28d
Weaning Post-Weaning
56d

30. Early Weaning Stress Induces Persistent
Disturbances in Intestinal Barrier Function
0 . 0 0 6
0 . 0 0 4
0 . 0 0 2
0 . 0 0 0
L a t e W e a n e d C o n t r o l ( L W C )
E a r l y W e a n i n g S t r e s s ( E W S )
***
W e a n i n g
*
1 2 8 5 6 1 1 2
T im e ( D a y s P o s t - w e a n i n g )
2
. h
3
H -m a n n i t o l f l u x ,  m o l . c m
Pohl et al (Manuscript in Progress)
* *

31. Experimental Design: Effects of Early Weaning Stress on
Long-Term Stress Responsiveness
Late Weaned Control (LWC)
Weaned at 28d
Early Weaning Stress
(EWS)
Weaned at 16 d
Mixing
Stress
Weaning Post-Weaning
54d
Barrier
function
measured
3hr post-stress

32. Early Weaning Stress Pigs Exhibit Exacerbated Intestinal
Injury Responses to Subsequent Production Stressors
0 . 2 0
0 . 1 5
0 . 1 0
0 . 0 5
0 . 0 0
2
. h
3
H -m a n n i t o l f l u x ,  m o l . c m
C o n t r o l
M i x i n g S t r e s s
*
E W S L W C
Moeser et al Gastroenterology, 2008
5
4
3
2
1
0
F e c a l S c o r e
C o n t r o l
M i x i n g S t r e s s
*
E W S L W C

33. Impact of Early Weaning Stress on Intestinal
Responses to Subsequent Infectious
Challenges

34. Early Weaning Stress Leads to Heightened Clinical
Disease in Response to Subsequent Enterotoxigenic E.
coli Challenge
Fecal Score
EWS Control
EWS + ETEC Challenge
LW Control
LW + ETEC Challenge
0 1 2 3 4
Days Post-ETEC Challenge
Fecal Score
4
3
2
1
0
McLamb et al., 2013 PLoS One. 8:e59838
Intestinal Permeability
0 . 0 1 0
0 . 0 0 8
0 . 0 0 6
0 . 0 0 4
0 . 0 0 2
0 . 0 0 0
2
. h
F D 4 f l u x , u g . c m
C o n t r o l
E T E C C h a l l e n g e
*
E W S L W C

35. 1 5 0 0 0
1 0 0 0 0
5 0 0 0
0
I L 8
p g /m g p r o t e i n
C o n t r o l
E T E C C h a l l e n g e
*
Early Weaning Stress Pigs Exhibit Suppressed Immune
Responses to Enterotoxigenic E. coli Challenge
5 0
4 0
3 0
2 0
1 0
0
p g /m g p r o t e i n
*
C o n t r o l
I L 6
E T E C C h a l l e n g e
4 0
3 0
2 0
1 0
0
# / h p f
N e u t r o p h i l s
C o n t r o l
E T E C C h a l l e n g e
*
EWS LWC EWS LWC EWS LWC
McLamb et al., 2013 PLoS One. 8:e59838

36. Day 4 post-challenge
16 d Weaning Age + ETEC
22 d Weaning Age + ETEC

37. Impact of Early Weaning Stress on Development
of Intestinal Epithelial and Immune Function
Long-term intestinal
epithelial barrier
dysfunction
Early Life Stress
Heightened stress
responsiveness
Exacerbated clinical
disease and intestinal
injury to subsequent
infectious challenge
Smith et al 2011. Am J Physiol-GIL
Moeser et al 2006. Am J Physiol-GIL
Moeser et al 2008. Am J Physiol-GIL

38. Early Weaning Stress is Characterized by Intestinal
Mast Cell Activation
EWS LWC
Smith et al. Am J Physiol Gastrointest Liver Physiol
2010;298:G352-G363

39. Mast Cells: Regulators of Homeostasis and Disease
Epithelial Barriers
Histamine
Nervous system cells
Proteases
TNF
Cytokines
Chemokines
Neuropeptides
Lipid Mediators
Immune cells
Endothelial cells
Endocrine cells
Muscle, Bone, Adipose
Stimulus

40. Mast Cells: Regulators of Homeostasis and Disease
Infectious Diarrheal Disease
Histamine
Proteases
TNF
Stimulus
Cytokines
Chemokines
Neuropeptides
Lipid Mediators
Cardiovascular disease
Infectious disease
pathogenesis
Allergy and Anaphylaxis
Immune Response
to Bacteria
Wound healing
Asthma/Allergic airway disease
Vaccine Immune
Responses
Bladder/Urogenital
disease
Sepsis/Endotoxemia
IBS
IBD
Food allergy
Autoimmune Disorders
GI Disease
Immune Response
to Viruses
Dermatitis

41. Stabilization of Mast Cells with Sodium Cromolyn Restores Intestinal
Barrier Function in Early Weaned Stress Pigs
a
L W C E W S E W S + C r o m o l y n
0 . 2 0
0 . 1 5
0 . 1 0
0 . 0 5
0 . 0 0
2
. h
3
H -m a n n i t o l f l u x ,  m o l . c m
Pohl et al (manuscript in preparation)

42. The Corticotropin Releasing Factor (CRF) System and the
Brain-Gut Axis
• CRF is a 41 aa peptide produced in the
hypothalamus and peripheral tissues
• Urocortins: Ucn 1, Ucn 2 and Ucn 3 are related
proteins that exert similar functions via CRF
receptor binding
• CRF coordinates many physiological functions
– HPA axis response
– Behavior: anxiety, coping, feed intake,
aggression
– GI effects Intestinal motor function, immune
cell activation, metabolism, inflammation
• CRF receptor system research focused on the
central and enteric nervous systems
– Behavior
– motility
• CRF system regulation of immune cells poorly
understood
Front. Psychiatry, 18 April 2011 | doi: 10.3389/fpsyt.2011.00016

43. Corticotropin Releaing Factor (CRF) Signaling is Up-regulated in
EWS Pig Intestine and is a Central Component of Intestinal Barrier
Function Regulation
0 . 0 5
0 . 0 4
0 . 0 3
0 . 0 2
0 . 0 1
Smith et al. Am J Physiol Gastrointest Liver 2011
L W C E W S
1 5
1 0
5
0
p g /m g p r o t e i n
L W C
E W S
*
Intestinal Permeability
0 . 0 0
2
. h
3
H -m a n n i t o l f l u x ,  m o l . c m
E W S
E W S +  - h e l i c a l C R F ( 9 - 4 1 )
*
Mucosal CRF levels
Mucosal CRF receptor expression

44. Blockade of CRF Receptors Prevented Mast Cell
Degranulation In Early Weaned Pig Intestine
Early Weaning
Stress
Weaning
Stress
+ CRF
receptor
Antagonist
Unstimulated
Mast Cell
(Unweaned)

45. Ex Vivo Approach to Study the CRF-Induced Mast Cell
Degranulation in the Porcine Intestine
Intestinal
Tissue
TER
Ohm’s law: V= IR
mV = μA * Ω
FITC-Dextran
CRF agonists

46. CRF Receptor Activation in the Porcine Intestine Induces
Mast Cell Activation and Intestinal Permeability
Overman et al 2013, PLoS One

47. Impact of Early Life Intestinal Stress the Development
of Intestinal Epithelial and Immune Function in the Pig
Long-term intestinal
epithelial barrier
dysfunction
Heightened stress
responsiveness
Exacerbated clinical
disease and intestinal
injury to subsequent
infectious challenge
Mast Cell
Degranulation
Activation of intestinal CRF system
Early Life Intestinal Stress (Early Weaning, infections)

48. Pathogenesis of Disease: Interactions Between
Pathogens, Host, and Environment
Pathogen
Environment
Susceptible
Hosts

49. Acknowledgements
• Boehringer Ingelheim Vetmedica, Inc.
Intestinal Stress Physiology Lab Funding
 American Gastroenterological Association
 National Institutes of Health (NIH) K08
DK084313 (AJM)
 NIH R01 HD072968 (AJM)
 NIH R03 DK097462 (AJM)
 National Pork Board
 NC Pork Council
 USDA
 Susan D’Costa PhD
 Saru Ayyadurai, Post Doc
 Susan D’Costa PhD, Research Associate
 Laura Edwards RLATG
 Liz Lennon, DVM DACVIM
 Julia Medland PhD student
 Emily Mackey, DVM/PhD student
 Brittney McLamb, DVM student
 Beth Overman PhD
 Calvin Pohl DVM, PhD student
 Laura Sommerville, Post Doc
 Janessa Winston, DVM, PhD student