Characterizing Intestinal Inflammation in Fanconi

Anemia Mutants

Characterizing Intestinal Inflammation in Fanconi

Anemia Mutants

Sam CarpentierUniversity of Oregon SPUR

2009

Sam CarpentierUniversity of Oregon SPUR

2009http://blogs.nature.com/nm/spoonful/zebrafish.jpg

Zebrafish Models of Intestinal

Inflammation

Zebrafish Models of Intestinal

Inflammation •Intestinal Alkaline Phosphatase Mutant

•Unable to detoxify LPS from Gram-negative bacteria.

•Sox 10 Mutant

•Defective gut peristalsis that leads to increased intestinal inflammation.

Why Fanconi Anemia?Why Fanconi Anemia? Fanconi Anemia is a DNA-repair disease that leads to problems such as cancer and blood-related diseases.

Fanconi Anemia is a DNA-repair disease that leads to problems such as cancer and blood-related diseases.

http://wpcontent.answers.com/wikipedia/commons/thumb/7/78/Fanconi%27s_anemia_101.jpg/190pxFanconi%27s_anemia_101.jpg

Inflammatory Bowel Disease Symptoms have been associated with some cases of Fanconi Anemia.

AimAim

Test the hypothesis that Fanconi mutants have increased intestinal inflammation.

Test the hypothesis that Fanconi mutants have increased intestinal inflammation.

We will count Myeloperoxidase (MPO) positive neutrophils in the zebrafish intestine.

We will count Myeloperoxidase (MPO) positive neutrophils in the zebrafish intestine.

Assay

Wild-Type Mutant

Fanconi D1 Mutants Do Not Have Elevated Inflammation

Fanconi D1 Mutants Do Not Have Elevated Inflammation

Data from 2 Independent trials

p=.64

WT FancD1 Mutant0

5

10

15

20

MPO Positive Cells Per Fish

Fanconi G Mutants Do Not Have Increased Inflammation

Fanconi G Mutants Do Not Have Increased Inflammation

p= .40

WT Fanc G Mutant0

5

10

15

20

25

MPO Positive Cells Per Fish

Fanconi C Mutants May Have Increased Inflammation

Fanconi C Mutants May Have Increased Inflammation

0123456789

101112

MPO Positive Cells Per Fish

WT

1

WT

2

Mu

t 2

Mu

t 1

p=.007

p=.70

Modifications of MPO Counting Protocol

Modifications of MPO Counting Protocol

• MPO-positive cells were counted that may have been outside of the gut.

•Further studies quantified the MPO-positive cells in sectioned tissue.

Fanconi C Mutants May Have Increased Inflammation

Fanconi C Mutants May Have Increased Inflammation

P=.18

WT FanC C Mutant0

5

10

15

20

MPO Positive Cells Per Fish

Fanc G Appear to Have Increased InflammationFanc G Appear to Have Increased Inflammation

P=.05

WT FancG Mut0

5

10

15

20

MPO Positive Cells Per Fish

Why is There Increased Inflammation in the Fanconi C Mutants?

Why is There Increased Inflammation in the Fanconi C Mutants?

•Immune imbalance, independent of microbiota.

•Increased inflammatory response to the microbiota.

inflamed mutant

derive germ-free

?

Raise Fanc C Mutants Germ- Free

(Without Microbiota)

Fanc C Mutant Inflammation is a Response to the

Microbiota

Fanc C Mutant Inflammation is a Response to the

Microbiota CV WT

Fanc C

CV Mut

GF WT

Fanc C

GF Mut

0

5

10

15

20

MPO Positive Cells Per Fish

WT p=.0003

Mutant p=.0001

Do Fanc C Mutants Exhibit Defective Peristalsis and

Bacterial Imbalance?

Do Fanc C Mutants Exhibit Defective Peristalsis and

Bacterial Imbalance?

5dpf 6dpf 7dpf 8dpf

Fanc C fish were fed fluorescent green food for three days, followed by a fluorescent red food.

Fanc C WT MovieFanc C WT Movie

QuickTime™ and aH.264 decompressor

are needed to see this picture.

Fanc C Mutant MovieFanc C Mutant Movie

QuickTime™ and aH.264 decompressor

are needed to see this picture.

ConclusionsConclusions There does not appear to be increased intestinal inflammation in the Fanc D1.

Intestinal Inflammation may be increased in both the Fanc C and G mutant fish.

Intestinal inflammation in the Fanc C mutants is a response to the microbiota.

Gut motility appears to be unimpaired in the Fanc C mutant fish.

There does not appear to be increased intestinal inflammation in the Fanc D1.

Intestinal Inflammation may be increased in both the Fanc C and G mutant fish.

Intestinal inflammation in the Fanc C mutants is a response to the microbiota.

Gut motility appears to be unimpaired in the Fanc C mutant fish.

Future DirectionsFuture Directions

Gut Microbiota Reciprocal Transplantation

Transfer CV Fanc C Mutant microbiota into GF Wild-Type Fish

Gut Microbiota Reciprocal Transplantation

Transfer CV Fanc C Mutant microbiota into GF Wild-Type Fish

inflamed mutantdonor

AcknowledgementsAcknowledgements

Erika MittgeKaren Guillemin Kate WoronowiczKristin Alligood Judith Eisen

The Guillemin Lab Peter O’Day Chelsie Fish SPUR Program Catharine WilsonTom Titus Poh Kheng Loi Rose UO Zebrafish Facility