SP-B Detection and Gene Expression in Chronic Rhinosinusitis

1
SP-B Detection and Gene Expression in Chronic
Rhinosinusitis

• Bradford A. Woodworth, MD
• Noam A. Cohen, MD, PhD
• Rachel Wood, BS
• Geeta Bhargave, BS
• John E. Baatz, PhD
• Rodney J. Schlosser, MD
• Department of Otorhinolaryngology HNS
• University of Pennsylvania Health System
• Medical University of South Carolina

2
Grant support

• Cystic Fibrosis Foundation

3
Surfactant

• Secreted in lungs by type II pneumocytes and
  Clara cells
• Phospholipids (lamellar bodies) 80-90
• Used for premature infants
• Decrease surface tension
• Decrease viscosity of mucus
• Proteins 10-15
• SP B and C hydrophobic, PL processing
  trafficking, anti-microbial properties
• SP A and D hydrophilic, immune functions

4
Surfactant in Airway Mucus

• Coats surface of gel layer to reduce surface
  tension
• Decreases the viscosity of mucus
• Increases mucociliary clearance
• SP-B facilitates properties of surfactant and is
  also shown to have direct anti-microbial
  properties

5
SP-B

• Extremely hydrophobic protein with multiple
  post-translational modifications
• SP-A and D found at mucosal and epithelial
  surfaces throughout the body
• SP-B originally thought to be limited to the
  lungs
• Recent studies show expression in the Eustachian
  tube mucosa

6
SP-B in Pulmonary Surfactant
SP-B
LB
Tubular Myelin
What role does surfactant play in the sinuses?
7
Prior Studies

• Phospholipid lamellar bodies in sinonasal
  epithelium

8
Prior Studies

• Hydrophilic surfactant proteins A and D in sinus
  mucosa
• Localize to epithelium and submucosal glandular
  elements
• Upregulated in cystic fibrosis CRS mucosa

Is there a role for SP-B?
9
Hypothesis

• SP-B is present in sinonasal mucosa and
  expression is altered in several types of CRS
  when compared to healthy controls.

10
Methods

• Sinus mucosal biopsies
• Allergic Fungal Rhinosinusitis (n7)
• Cystic Fibrosis (n 4)
• Non-atopic CRS with nasal polyps (n5)
• Healthy controls (n5)
• Quantitative RT-PCR, immunoblot,
  immunohistochemistry

11
Methods Cycle threshold (Ct)
12
Methods Cycle threshold (Ct)

• Delta Ct (?Ct) - Ct for mRNA subtracted from Ct
  of internal control (18s rRNA).
• Eliminates effect of differences in sample
  concentration on Ct.
• Individual ?Ct values of each subtype of CRS are
  compared to the healthy control tissue

13
SP-B Quantitative RT-PCR

p 0.004
167-fold elevation in CF patients compared to
healthy controls
14
Detection of SP-B Proprotein
42 kDa
Detection of the proprotein and intermediate
forms confirms translated product

• Where is the protein produced and secreted?

15
Immunofluorescence
Sinus Epithelium
Submucosal Glands
SP-B expression green, Nuclear stain in blue
SP-B localizes to the epithelium and submucosal
glands
16
Discussion

• SP-B was significantly upregulated in CF CRS
  mucosa
• Pseudomonas produces proteases known to degrade
  SPs (Malloy et al).
• SP-B upregulated in response to degradation of
  SP-B and surfactant by Pseudomonas.
• Alternatively, increased submucosal glands in CF
  mucosa contribute more mRNA transcripts to sample

17
Conclusion

• SP-B is upregulated in cystic fibrosis CRS and is
  produced by the epithelium and submucosal glands
  of the sinonasal cavities.
• Further studies indicated to investigate the role
  that SP-B and surfactant have in CRS.

18
Future directions

• Anti-microbial properties of SP-B as a potential
  therapy for infectious CRS
• In vivo and in vitro models
• Further delineation of protein expression and
  specific localization with immunoelectron
  microscopy

19
Thank You