Oxford Inflammatory Bowel Disease MasterClass Microbial-immune interaction Dr Satish Keshav, Oxford, UK
Oxford Inflammatory Bowel Disease MasterClass
Microbial-immune interaction
Dr Satish Keshav, Oxford, UK
Microbial-Immune Interaction
Dr Satish Keshav DPhil FRCP
John Radcliffe Hospital
Oxford MasterClass 2013
Wadham College
Session 1: The Causes of IBD
‘‘I can only regret that the etiology of the condition remains in obscurity, but I trust that ere long further consideration will clear up the difficulty.’’
Dalziel T K (1913) Chronic interstitial enteritis. Br Med J 2:1068–70
Ulcerative colitis & Crohn’s
Ulcerative colitis & Crohn’s
Manifestations of IBD
Antibiotics
Probiotics
Elemental diet
Corticosteroids
Thiopurines
Anti-TNF mAbs
Investigational drugs
5-ASA / Mesalamine derivatives
Stomach
Oesophagus
Transverse colon
Jejunum
Sigmoid colon
Rectum
Ascending colon
Terminal ileum
Appendix
Liver
<103
organisms/ml
>1011
organisms/ml
107-108
organisms/ml
>500 bacterial species
Commensals & potential
pathogens
Incl. Firmicutes
Bacteroidetes
Proteobacteria
Actinobacteria
Bacteria are Relevant to IBD
1. Experimental models & commensal bacteria 1. Germ-free vs Gnotobiotic vs SPF 2. Transferable colitis models 3. Induce specific immune responses – REGIIIγ, IgA, IEL, Treg, Th17
2. NOD2 interacts with bacterial muramyldipeptide
3. Evidence from IBD
Experimental evidence
Hooper, Littman, & MacPherson (2012) Science vol 336, pp 1268
Honda & Littman (2012) Annual Review of Immunology vol 30, pp 759
Blumberg & Powrie (2012) Science Translational Medicine vol 4, pp 1
Clinical evidence:
infection can trigger IBD
Rodriguez, Ruigomez & Panes (2006) Gastroenterology vol 130, pp 1588
Porter et al (2008) Gastroenterology vol 135, pp 781
Ternhag et al (2008) Emerging Infectious Disease vol 14, pp 143
Clinical evidence
Efficacy of E. coli Nissl
AIEC in Crohn’s
Metronidazole post-operative Crohn’s
Etc..
Paneth cells
Paneth J (1888) Arch Mikro Anat 31, 113-191
Joseph Paneth 1857-1890
No IL-1β, IL-6, MCP-1 RNA
No TNF protein
Keshav et al (1990) J Exp Med 171, 327-32
TNF positive Negative control
Paneth cells Paneth cells
TNF in murine intestine
TNFΔARE mice over-express TNFα
Translation of the TNFα mRNA into protein is blocked by AU-rich elements (ARE) in the 3’ untranslated region of the gene
Deletion of the ARE in transgenic mice
increases TNFα protein production in leukocytes and other cells
Kontoyiannis et al (1999) Immunity. 10, 387-398
Crohn’s-like ileitis in TNFΔARE
Lala et al (2003) Gastroenterology 125, 47-57
NOD2 is constitutively expressed
in Paneth cells
NOD2 protein interacts with MDP
3 common mutations associated with Crohn’s disease
Constitutively expressed in circulating monocytes
Altered signalling in response to MDP
Ogura et al (2001) Nature
41, 603
Hugot et al (2001) Nature
41, 599
Hampe et al (2001) Lancet
357, 1925
NOD2 & ileal Crohn’s
Disease location +/+ +/- -/- -/*- n
Control 84 15 0.3 0.3 349
Crohn's total 61 27 2.9 9 244
Ileal Crohn's 56 30 3.5 11 201
No ileal disease 83 16 0 0 43
Ahmad et al (2002) Gastroenterology 202, 854-66
Allele
frequency
8.1%
25.4%
29.5%
8%
NOD2 & ileal Crohn’s
Disease location +/+ +/- -/- -/*- n
Control 84 15 0.3 0.3 349
Crohn's total 61 27 2.9 9 244
Ileal Crohn's 56 30 3.5 11 201
No ileal disease 83 16 0 0 43
Ahmad et al (2002) Gastroenterology 202, 854-66
Allele
frequency
8.1%
25.4%
29.5%
8%
Wehkamp et al (2004)
Gut 53, 1658
Defensins ?
Crohn's disease: a
defensin deficiency
syndrome?
Simms et al (2008) Gut, 57:903-10
Inflammation and -Defensins
NOD2-/- mice + H. hepaticus
Biswas et al (2010) PNAS 107:14739
NOD2-/- + H. hepaticus + HD5
Biswas et al (2010) PNAS 107:14739
Symbiotic bacteria induce RegIIIγ in Paneth cells
Cash et al (2006) Science, 313:1126-30
RegIIIγ Regulates Microbial Distance in the Gut
Vaishnava et al (2011) Science, 334:255
Autophagy-related genes: ATG16L & IRGM
Parks et al (2007) Nat Genet, 39:830-2
Cadwell et al (2008) Nature, 456:259-63
ATG16L mutation affects the morphology of Paneth cells
-catenin & TCF4 regulate expression of α-defensins
van Es et al (2005) Nat Cell Biol, 7:381-6
NOD2 variation: Morphological effect
Wild typeNOD2
-/-0
5
10
15
20
* p<0.005
Hig
hes
t p
os
itio
nfr
om
cry
pt
ba
se
(me
an
of
10
cry
pts
)
Wild typeNOD2
-/-0
5
10
15
20
* p<0.005
Nu
mb
er
of
Pa
ne
thc
ells
pe
r c
ryp
t(m
ea
n o
f 1
0 c
ryp
ts)
Abeya & Keshav, unpublished data
Effect of NOD2 on cell position
Similar to EphB3 knockout mouse
EphB3 - major wnt/-catenin target
Batlle et al (2002) Cell 111,
251-63
Clevers, H et al (2012) NEJM, 367:1560
Paneth cells Alter Intestinal Stem Cell Function
Paneth cells in
IBD
Paneth cells in
IBD
Paneth cells in
IBD
Fecal Diversion
Emanoel Lee (1975) Ann R Coll Surg vol 59
Background
First reported 1913 for "physiological rest“
Early response up to 86%
Continuity of bowel re-established in 22%
Oberhelman HA, Kohatsu S, Taylor KB, Kivel RM. Diverting ileostomy in the surgical
management of anal and perianal Crohn’s disease of the colon. Am J Surg 1968;115:231–240.
Burman JM, Thompson M, Cooke WT, Alexander-Williams J. The effects of diversion of intestinal contents on the progress of Crohn’s disease of the large bowel. Gut 1971;2:11.
Frizelle FA, Santoro GA, Pemberton JH. The management of perianal Crohn’s disease. Int J Colorectal Dis 1996;11:227–237.
McClane, Rombeau JL. Anorectal Crohn’s disease. Surg Clin North Am 2001;81:169–183.
Emanoel Lee (1975) Ann R Coll Surg vol 59
Early Response = (14+22)/46=78%
Early Response = (14+22)/46=78%
Late Response = 9/14=64%
Oxford Experience 1970-1997
Edwards et al (2000) Br J Surg 87: 1063
Oxford Experience 1970-1997
Edwards et al (2000) Br J Surg 87: 1063
Early Response = 48/55=87%
Late Response = 11/25=44%
Early Response = 15/18=83%
Late Response = 2/15=13%
Or
2/4=50%
Leuven Experience
Contact with intestinal fluids for 8 days: focal infiltration of
mononuclear cells, eosinophils, and polymorphonuclear cells in the lamina propria, small vessels, and epithelium
Increased Epithelial HLA-DR, Mononuclear cell RFD-7, RFD-9, ICAM-1, LFA-1
Dilatation of ER and Golgi apparatus in epithelial cells
D'Haens GR, Geboes K, Peeters M, Baert F, Penninckx F, Rutgeerts P. Gastroenterology 1998 114(2):262-7.
Oxford Experience after 1997
60 Patients
Average age: 34 years (12-85)
Sex: F:M 23:34
Indication for defunctioning
Peri-anal Crohn’s Disease: 34 (60%)
Crohn’s Colitis: 23 (40%)
Oxford Experience after 1997
Early response in 42%
Intestinal continuity re-established in 19 % Marc Martí-Gallostra et al Unpublished
Monica Cesarini – Unpublished data
Microbial-Immune Interaction as a cause of IBD
Compelling Evidence from Experimental Models
Suggestive Clinical Evidence
Genetic
Epidemiological
Pathological
Need for Human Models of Microbial-Immune Interaction