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