1. The Role of Wfdc1 in the
Inflammatory Response to
Influenza Virus Infection
Adenuga Gbadebo Jr.
Laboratory of Dr. David Rowley
Mentor: Steven Ressler, Ph.D.

2. Wfdc1 gene is a member of
the WAP family and
expresses the ps20 protein
• secreted proteins
• most members have
serine protease
inhibitor function
• Anti-inflammatory
• Anti-microbial
Typical structure of WAP motifTypical structure of WAP motif
Structural model shows typical WAP four disulphide core domain from elafin protein.Structural model shows typical WAP four disulphide core domain from elafin protein. YellowYellow=four disulphide=four disulphide
bridges formed by eight cysteine residues.bridges formed by eight cysteine residues. Blue arrowsBlue arrows= strands.= strands. RedRed=proteinase-binding loop of elafin. Courtesy=proteinase-binding loop of elafin. Courtesy
of Alain Doucet, CREFSIP and Department of Biochemistry and Microbiology, Laval University, Quebec, Canada.of Alain Doucet, CREFSIP and Department of Biochemistry and Microbiology, Laval University, Quebec, Canada.

3. Wfdc1 knock out knock in
mouse
9.5kb
6.5kb
11 22 33 44 6655
Bgal------PGKNeo
βgal------PGKNeo
ATG
RV
9.5kb w.t.
allele
6.5kb knockout-in allele
77
1.3kb Probe
Wfdc1 knock out knock in
mouse

4. Influenza infection of Wfdc1
Het and null mice
Day 4 Influenza titers
ps20 +/- ps20 -/-
0
1
2
3
4
5
6
7
3.2
5.8
Virus titers are higher in the presence of ps20
Genotype of infected mice
Titer(log10)
p=0.0013p=0.0013
n=15n=15

5. Purpose
• To determine if the difference in
Influenza viral infectivity in the Wfdc1
null mouse is due to alterations in
inflammatory cell infiltration

6. Two main types of
inflammatory cells
Neutrophils
•Recruited from blood
•Involved in bacterial phagocy
•Short lived
Macrophage
•Tissue fixed
(alveolar
macrophage)
•Phagocytise
infected cells and

7. Experiments
• To stain for β-gal as a surrogate marker for Wfdc1
expression in mice
• To detect macrophages in influenza infected lungs
of the w.t., Het, and null Wfdc1 mice
• To detect neutrophils in influenza infected lungs of
the w.t., Het, and null Wfdc1 mice

8. Procedure
• Immunohistochemistry on influenza infected lungs from
mice with 2 copies of the Wfdc1 gene (w.t.), 1 copy of
the Wfdc1 gene and 1 copy of the B-gal gene (Het) , and
no copies of the Wfdc1 gene and two copies of the B-gal
gene (Null)
tis
sue
1° Ab
β-gal
Neutrophil
Macrophage
Streptavidin
Alkaline Phosphate
Biotin 2°Ab
Vector Red
substrate

9. β-gal is expressed in smooth
muscle of both the Het and
Null Wfdc1 mice and not in
w.t. mice
w.t. Het Null
vessel
bronchiole
vessel
bronchiole

10. An Increase in Macrophage staining
in Influenza infected null versus
w.t., Het, Wfdc1 mice lungs
Nullw.t.Het

11. No difference in neutrophil
staining in Influenza infected
Null versus Het mouse lungs
HetNull

12. Conclusions
• By staining for β-gal as a surrogate marker for Wfdc1
expression we have shown that expression in the mouse lung
is specific for smooth muscle surrounding small vessels and
bronchioles
• This result corroborates published staining for Wfdc1 in rat
and human tissue
• An increase in Macrophage staining was shown in Null infected lungs
versus both Het and w.t. lungs
• This result could explain the observation that the Null mice are more
resistant to influenza infection because an increase in macrophages
correlates with viral resistance.
• Similar staining for Neutrophils was shown in both Null and Het infected
lungs
• Neutrophils are associated with a more chronic inflammatory state and
usually with bacterial clearance

13. Acknowledgements
• David Rowley, Ph.D.
• Steven Ressler, Ph.D.
• David Barron
• Truong Dang
• Feng Yang, Ph.D.
• Danielle Dory
• Woosook Kim
Rowley LabRowley Lab