Adam Moeser DVM PhD

Associate Professor of Gastrointestinal Biology & Swine Medicine

Department of Population Health & Pathobiology

Gut Physiology from a Pathogens Point of View/Interactions

Pathogenesis of Disease: Interactions Between Pathogens, Host, and Environment

Pathogen

Environment

SusceptibleHosts

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

Intestinal Barrier Function

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-, HCO3

-, and water secretion from the crypts: e.g. E. coli, S. Typhimurium, etc

HCO3-

HCO3-

Circulation

Tight Junctions

Extrinsic Barrier

Microbiota

Immune System

Nutrients Pathogens ToxinsH20

Mucus Antimicrobial peptides

IgA

Immunologic BarrierIntrinsic Barrier

EpithelialCells

Lamina Propria

IntestinalLumen

Cl-

The Gastrointestinal Barrier

Enteric nervous system

Mechanisms of Intestinal Secretion

ETEC Rotavirus

Bacterial toxins and Inflammatory products

Moeser AJ and Blikslager, JAVMA 231:56-67, 2007

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

Mechanisms of Intestinal Absorption

Moeser AJ and Blikslager, JAVMA 231:56-67, 2007

ETEC

Rotavirus

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

Cl-

Villous Atrophy

Mucosal surface area

Nutrient and water absorption

Diarrhea

H2O

Sanford

Control

Coccidiosis

Inflammatory Diarrhea: Salmonella Typhimurium

Cl-

H20

INFLAMMATION

HCO3-

HYPERSECRETION

IL1-βIL-8

PGE2

Cl-

H20

HCO3-

MALABSORPTIONProgression toSubacute/Chronic lesions

Recruitment of neutrophils

S typhimurium

Salmonella Enteritis

Healthy Control Salmonella Typhimurium enteritis

Pathogenesis of Disease: Interactions Between Pathogens, Host, and Environment

Pathogen

Environment

SusceptibleHosts

Stress

Stress is a Major factor in the Onset and Exacerbation of GI Disease in Animals and People

Stress

http://www.stress-management-for-health.com/physical-effects-of-stress.html

Stress

Functional and/or inflammatory GI Disorders

Diarrhea

Susceptibility to GI Infections

Poor weight gain/feed conversion

The biological mechanisms by which stress causes disease remain poorly understood

Hyper-activated or Suppressed Immune response

Intestinal Stress Physiology Lab• Long-Term Goal: Understand the biological

mechanisms by which stressors impact gut health

Weaning is the Most Profound Stress a Pig Encounters in Production

Weaning Stressors

Maternal separation

Change in environment

Increased exposure to pathogens

Fighting and establishment

of social hierarchy

Abrupt transition in diet

Transportationstress

Post-Weaning Health Challenges in the Pig

How does production stress impact gut defense and disease susceptibility?

HCO3-

Circulation

Tight Junctions

Extrinsic Barrier

Microbiota

Immune System

Nutrients Pathogens ToxinsH20

Mucus Antimicrobial peptides

IgA

Immunologic BarrierIntrinsic Barrier

EpithelialCells

Lamina Propria

IntestinalLumen

Cl-

The Gastrointestinal Barrier

Enteric nervous system

HCO3-

Nutrients Pathogens Toxins

H20

EpithelialCells

Lamina Propria

IntestinalLumen Cl-

INFLAMMATION

Fluid Loss

Compromised Barrier Under Stress: “Leaky Gut”

IMMUNOSUPPRESSIONAcuteStages Chronic stages

HCO3-

Circulation

Tight Junctions

Extrinsic Barrier

Microbiota

Immune System

Nutrients Pathogens ToxinsH20

Mucus Antimicrobial peptides

IgA

Immunologic BarrierIntrinsic Barrier

EpithelialCells

Lamina Propria

IntestinalLumen

Cl-

The Gastrointestinal Barrier

Enteric nervous system

HCO3-

Nutrients Pathogens Toxins

H20

EpithelialCells

Lamina Propria

IntestinalLumen Cl-

INFLAMMATION

Fluid Loss

Compromised Barrier Under Stress: “Leaky Gut”

IMMUNOSUPPRESSIONAcuteStages Chronic stages

Measurement of Intestinal Epithelial Barrier Function: Ussing Chamber Technique

Intestinal Tissue

TEROhm’s law: V= IR

mV = μA * Ω

FITC-Dextran Supernatant3H-Mannitol14C-Inulin

Increased Flux = compromised gut barrierDecreased flux = Intact gut barrier

Weaning Stress Breaks Down Intestinal Barrier Function

Moeser et al. Am J Physiol Gastrointest Liver Physiol. 292:G173-81

0

20

40

60

80

100

TE

R,

.cm

2

UnweanedWeaned

*

0.00

0.02

0.04

0.06

3H

-man

nit

ol f

lux,

mo

l.cm

2.h *

UnweanedWeaned

Weaning

Increased Intestinal Permeability

Influence of Chronic Production Stressors on Intestinal Barrier Function in Pigs

Control Mixing/crowding Stress

Control (TN) Heat Stress

Mixing and Crowding Stress Chronic Heat Stress

P<0.01 P<0.01

Influence of Chronic Production Stressors on Intestinal Glucose Transport

Control (TN) Heat Stress

Mixing and Crowding Stress Chronic Heat Stress

∆Is

c, u

A/c

m2

∆Is

c, u

A/c

m2

Mixing/crowding Stress

P<0.01 P<0.01

Control

Influence of Pig Age on Weaning-induced Intestinal Injury

Delayed Weaning Ameliorates Weaning-Induced Intestinal Barrier Dysfunction

Moeser et al. Am J Physiol Gastrointest Liver

Physiol. 293:G413-21

0.00

0.01

0.02

0.03

0.04

0.05

3H

-man

nit

ol f

lux,

mo

l.cm

2.h *

UnweanedWeaning stress

EWS(16 d weaning)

LWC(28 d weaning)

15 18 21 23 280.00

0.02

0.04

0.06

0.08

3H

-man

nit

ol f

lux,

mo

l.cm

2.h

Weaning Age

Smith et al. Am J Physiol Gastrointest Liver Physiol 2010;298:G352-G363

Experimental Design: Long-term Impact of Weaning Age on Intestinal Barrier Function

112d

Early Weaning Stress (EWS)

Weaned at 16 d

Late Weaned Control (LWC) Weaned at 28d

Intestinal permeability measurements

1d 28d

Post-WeaningWeaning

56d

Early Weaning Stress Induces Persistent Disturbances in Intestinal Barrier Function

Pohl et al (Manuscript in Progress)

0.000

0.002

0.004

0.006

Time (Days Post-weaning)

3H

-man

nit

ol f

lux,

mo

l.cm

2.h

Late Weaned Control (LWC)Early Weaning Stress (EWS)

Weaning

28 56 1121

***

* * *

Experimental Design: Effects of Early Weaning Stress on Long-Term Stress Responsiveness

Early Weaning Stress (EWS)

Weaned at 16 d

Late Weaned Control (LWC) Weaned at 28d

Mixing Stress

Post-WeaningWeaning

54d

Barrier function measure

d 3hr post-stress

Early Weaning Stress Pigs Exhibit Exacerbated Intestinal Injury Responses to Subsequent Production Stressors

Moeser et al Gastroenterology, 2008

0.00

0.05

0.10

0.15

0.20

3H

-man

nit

ol f

lux,

mo

l.cm

2.h *

EWS LWC

ControlMixing Stress

0

1

2

3

4

5

Fec

al S

core

*

EWS LWC

ControlMixing Stress

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

Early Weaning Stress Leads to Heightened Clinical Disease in Response to Subsequent Enterotoxigenic E. coli Challenge

McLamb et al., 2013 PLoS One. 8:e59838

Days Post-ETEC Challenge0 1 2 3 4

Fec

al S

core

0

1

2

3

4

EWS Control

EWS + ETEC Challenge

LW Control

LW + ETEC Challenge

Fecal Score Intestinal Permeability

0.000

0.002

0.004

0.006

0.008

0.010

FD

4 fl

ux,

ug

.cm

2.h

*

EWS LWC

ControlETEC Challenge

0

5000

10000

15000

IL8

pg

/mg

pro

tein

ControlETEC Challenge

*

Early Weaning Stress Pigs Exhibit Suppressed Immune Responses to Enterotoxigenic E. coli Challenge

0

10

20

30

40

50p

g/m

g p

rote

in*

ControlETEC Challenge

IL6

0

10

20

30

40

#/h

pf

ControlETEC Challenge

Neutrophils

*

EWS LWC EWS LWC EWS LWC

McLamb et al., 2013 PLoS One. 8:e59838

16 d Weaning Age + ETEC

Day 4 post-challenge

22 d Weaning Age + ETEC

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

Early Weaning Stress is Characterized by Intestinal Mast Cell Activation

Smith et al. Am J Physiol Gastrointest Liver Physiol 2010;298:G352-G363

EWS LWC

Histamine

Proteases

TNF

Cytokines

Chemokines

Neuropeptides

Lipid Mediators

Epithelial Barriers

Immune cells

Nervous system cells

Endothelial cells

Muscle, Bone, Adipose

Endocrine cells

Stimulus

Mast Cells: Regulators of Homeostasis and Disease

Mast Cells: Regulators of Homeostasis and Disease

Histamine

Proteases

TNF

Cytokines

Chemokines

Neuropeptides

Lipid Mediators

Stimulus

Infectious disease pathogenesis

Cardiovascular disease

Allergy and Anaphylaxis

Immune Response to Bacteria

Wound healingAsthma/Allergic airway disease

Vaccine Immune Responses

Bladder/Urogenital disease

Sepsis/Endotoxemia

IBS

Infectious Diarrheal Disease

IBD

Food allergy

Autoimmune Disorders

GI Disease

Immune Response to Viruses

Dermatitis

Stabilization of Mast Cells with Sodium Cromolyn Restores Intestinal Barrier Function in Early Weaned Stress Pigs

Pohl et al (manuscript in preparation)

LWC EWS EWS + Cromolyn0.00

0.05

0.10

0.15

0.20

3H

-man

nit

ol f

lux,

mo

l.cm

2.h

a

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

Corticotropin Releaing Factor (CRF) Signaling is Up-regulated in EWS Pig Intestine and is a Central Component of Intestinal Barrier Function Regulation

Smith et al. Am J Physiol Gastrointest Liver 2011

LWC EWS0

5

10

15

pg

/mg

pro

tein

LWCEWS

*Intestinal Permeability

0.00

0.01

0.02

0.03

0.04

0.05

3H

-man

nit

ol f

lux,

mo

l.cm

2.h

EWSEWS +-helical CRF(9-41)

*

Mucosal CRF levels

Mucosal CRF receptor expression

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)

Ex Vivo Approach to Study the CRF-Induced Mast Cell Degranulation in the Porcine Intestine

Intestinal Tissue

TEROhm’s law: V= IR

mV = μA * Ω

FITC-Dextran

CRF agonists

CRF Receptor Activation in the Porcine Intestine Induces Mast Cell Activation and Intestinal Permeability

Overman et al 2013, PLoS One

Impact of Early Life Intestinal Stress the Development of Intestinal Epithelial and

Immune Function in the Pig

Long-term intestinal epithelial barrier

dysfunction

Early Life Intestinal Stress (Early Weaning, infections)

Heightened stress responsiveness

Exacerbated clinical disease and intestinal injury to subsequent infectious challenge

Mast Cell Degranulation

Activation of intestinal CRF system

Pathogenesis of Disease: Interactions Between Pathogens, Host, and Environment

Pathogen

Environment

SusceptibleHosts

Acknowledgements

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

Funding Intestinal Stress Physiology Lab

• Boehringer Ingelheim Vetmedica, Inc.