Paneth Cell Phenotype Correlates with Genetics, Transcriptome Profile, Pathologic Hallmark and Predicts Clinical Outcome in

Patients with Crohn's Disease

Ta-Chiang Liu, M.D., Ph.D.

Assistant Professor

Department of Pathology and Immunology

Washington University in St. Louis

Disclosures

Nothing to disclose

Goal: Identification of a cellular readout that synthesizes genetic and environmental

factors and subclassifies Crohn’s Disease

EnvironmentGenetic

Susceptibility

Cellular readout

Disease subclassification

Risk Loci Predicted to Affect Paneth Cell FunctionATG16L1NOD2XBP1ITLN1

Paneth Cells: Critical Roles in Innate Immunity

AntimicrobialsLysozymeα-Defensins

Secretion defect in Atg16L1-deficient Paneth cells

WT Atg16l1 deficient

Cadwell et al., Nature 2008.

NOD2 Variants and Disease Susceptibility

Common CD-associated variants

Rare CD-associated variants (6 known)

Allele carriage rate: <5%

Allele carriage rate: <1%

Relative risk for development of CD:NOD2 Heterozygous = 2-3NOD2 Homozygous = ~20

HypothesisNOD2 risk variants may be associated with an abnormal granule

phenotype in the Paneth cells of CD patients

Materials and Methods De-identified ileocolectomy tissue from CD patients Inclusion criteria:

Access to proximal margin of ileocolic resection Minimum of 100 well-oriented crypts No active or chronic inflammatory disease in the ileal

sections used for lysozyme stain

Scale bars: 10 µMImmunofluorescence

Lysozyme

Hoescht

Gastroenterology, in press

NOD2 Risk Variants are Associated with Abnormal Paneth cell Phenotype

diminished

NOD2 Risk Variants are Associated with Abnormal Paneth cell Phenotype

*p<0.05 compared to 0 risk variants

Gastroenterology, in press

NOD2 and ATG16L1 Risk Alleles are Additive for Abnormal Paneth Cell Phenotypes

Activated Immune Response Profile is Associated with Abnormal Paneth Cells

Phenotype

GO term analysis performed using DAVID Gastroenterology, in press

Paneth cell phenotype is associated with clinical outcome

Gastroenterology, in press

CD patients from WU and CSMC (n=143) underwent resection and received post-op prophylactic therapy

End point: endoscopic evidence of disease recurrence after surgery

Paneth cell phenotype as predictive cellular biomarker for

CD – practical issues

% Abormal Paneth cellsin uninvolved area

% A

bn

orm

al P

anet

h c

ells

in in

volv

ed a

rea

0 10 20 30 40 500

20

40

60

80

Paneth cell phenotypes in involved areas correlate with that of the uninvolved areas

P<0.0001

% Abormal Paneth cellsin first resection

% A

bn

orm

al P

anet

h c

ells

in s

eco

nd

res

ecti

on

0 10 20 30 40 500

10

20

30

40

Paneth cell phenotype remains stable over the years

(n=30)P<0.0001

Paneth cell defects may be patchy: how many crypts are needed to generate readout equivalent

to resection specimens?

“Virtual biopsy”

At least 40-50 crypts are needed to generate equivalent results as with resection specimens.

% Abormal Paneth cellsin resection

% A

bn

orm

al P

anet

h c

ells

in b

iop

sy

0 20 40 60 80 1000

10

20

30

40

50

Paneth cell phenotypes in matched biopsy and resection specimens are consistent

(n=20)P=0.0004

Normal

Disord

ered

Dimin

ished

Diffuse

Exclu

ded

Enlarg

ed0

20

40

60

80

100

To

tal P

an

eth

cel

ls (

%)

Normal

Disord

ered

Dimin

ished

Diffuse

Exclu

ded

Enlarg

ed0

20

40

60

80

100

To

tal P

an

eth

cel

ls (

%)

Bad Paneth cell phenotype is more prevalent in pediatric CD

Adult (n=124) Pediatric (n=77)

P< 0.0001

(Collaboration with Nita Salzman)

15%47%

Significant increase in bad Paneth cell phenotype over the past 30 years

1980-1982 (n=52)

Normal

Disord

ered

Dimin

ished

Diffuse

Exclu

ded

Enlarg

ed

0

20

40

60

80

100

Paneth Cell Phenotype

To

tal P

an

eth

ce

lls (

%)

P=0.0328

Normal

Disord

ered

Dimin

ished

Diffuse

Exclu

ded

Enlarg

ed0

20

40

60

80

100

To

tal P

an

eth

cel

ls (

%)

2011-2013 (n=124)

15%3%

1980-1982 (n=52)

Summary

• Bad Paneth cell phenotype is associated with CD-associated risk alleles, distinct gene expression profile, pathology hallmark, and clinical outcome.

• Paneth cell phenotype is spatially and temporally stable.

• Paneth cell phenotype analysis can be performed using routine biopsy material.

• Bad Paneth cell phenotype is commonly seen in pediatric patients, and appears to be a new form of disease that have emerged over the last 30 years.

AcknowledgementCedars-Sinai

Dalin LiFadi TowficNir ModianoRachel WinterTalin HaritunainsDeepti DhallStephan TarganDermot P. B. McGovern

Washington University Kelli VanDussen Robi D. Mitra Rich Head Rodney D. Newberry Feng Gao Thaddeus S. Stappenbeck

Harvard/MGH

Ramnik Xavier

Medical College of Wisconsin

Nita Salzman