dr. adam moeser - gut physiology from a pathogens point of view
DESCRIPTION
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-seminarTRANSCRIPT
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.