SP-B Detection and Gene Expression in SP-B Detection and Gene Expression in Chronic RhinosinusitisChronic Rhinosinusitis

Bradford A. Woodworth, MDBradford A. Woodworth, MDNoam A. Cohen, MD, PhDNoam A. Cohen, MD, PhD

Rachel Wood, BSRachel Wood, BSGeeta Bhargave, BSGeeta Bhargave, BSJohn E. Baatz, PhDJohn E. Baatz, PhD

Rodney J. Schlosser, MDRodney J. Schlosser, MD

Department of Otorhinolaryngology – HNSDepartment of Otorhinolaryngology – HNSUniversity of Pennsylvania Health SystemUniversity of Pennsylvania Health System & Medical University of South Carolina& Medical University of South Carolina

Grant supportGrant support

Cystic Fibrosis FoundationCystic Fibrosis Foundation

SurfactantSurfactant

Secreted in lungs by type II pneumocytes and Secreted in lungs by type II pneumocytes and Clara cellsClara cells

Phospholipids (lamellar bodies): 80-90%Phospholipids (lamellar bodies): 80-90%– Used for premature infantsUsed for premature infants– Decrease surface tensionDecrease surface tension– Decrease viscosity of mucusDecrease viscosity of mucus

Proteins: 10-15%Proteins: 10-15%

– SP BSP B and C: hydrophobic, PL processing & and C: hydrophobic, PL processing & trafficking, anti-microbial propertiestrafficking, anti-microbial properties

– SP A and D: hydrophilic, immune functionsSP A and D: hydrophilic, immune functions

Surfactant in Airway MucusSurfactant in Airway Mucus

Coats surface of gel layer to reduce surface tensionCoats surface of gel layer to reduce surface tension Decreases the viscosity of mucusDecreases the viscosity of mucus Increases mucociliary clearanceIncreases mucociliary clearance SP-BSP-B facilitates properties of surfactant and is also facilitates properties of surfactant and is also

shown to have direct anti-microbial propertiesshown to have direct anti-microbial properties

SP-BSP-B

Extremely hydrophobic protein with multiple Extremely hydrophobic protein with multiple post-translational modificationspost-translational modifications

SP-A and D found at mucosal and epithelial SP-A and D found at mucosal and epithelial surfaces throughout the bodysurfaces throughout the body

SP-B originally thought to be limited to the SP-B originally thought to be limited to the lungslungs

Recent studies show expression in the Recent studies show expression in the Eustachian tube mucosaEustachian tube mucosa

LB

Tubular Myelin

SP-B

SP-B in Pulmonary Surfactant

What role does surfactant play in the sinuses?

Prior StudiesPrior Studies

Phospholipid lamellar bodies in Phospholipid lamellar bodies in sinonasal epitheliumsinonasal epithelium

Prior StudiesPrior Studies

Is there a role for SP-B?

Hydrophilic surfactant Hydrophilic surfactant proteins A and D in sinus proteins A and D in sinus mucosamucosa– Localize to epithelium and Localize to epithelium and

submucosal glandular submucosal glandular elementselements

– Upregulated in cystic fibrosisUpregulated in cystic fibrosis CRS mucosaCRS mucosa

HypothesisHypothesis

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

MethodsMethods

Sinus mucosal biopsies Sinus mucosal biopsies – Allergic Fungal Rhinosinusitis (n=7)Allergic Fungal Rhinosinusitis (n=7)– Cystic Fibrosis (n = 4) Cystic Fibrosis (n = 4) – Non-atopic CRS with nasal polyps (n=5) Non-atopic CRS with nasal polyps (n=5) – Healthy controls (n=5)Healthy controls (n=5)– Quantitative RT-PCR, immunoblot, Quantitative RT-PCR, immunoblot,

immunohistochemistryimmunohistochemistry

Methods – Cycle threshold (Ct)Methods – Cycle threshold (Ct)

Delta Ct (∆Ct) - Ct for mRNA Delta Ct (∆Ct) - Ct for mRNA subtracted from Ct of internal subtracted from Ct of internal control (18s rRNA).control (18s rRNA).– Eliminates effect of differences in Eliminates effect of differences in

sample concentration on Ct.sample concentration on Ct. Individual ∆Ct values of each Individual ∆Ct values of each

subtype of CRS are compared to subtype of CRS are compared to the healthy control tissuethe healthy control tissue

Methods – Cycle threshold (Ct)Methods – Cycle threshold (Ct)

SP-B Quantitative RT-PCRSP-B Quantitative RT-PCR

* p = 0.004

167-fold elevation in CF patients compared to healthy controls

*

Detection of SP-B ProproteinDetection of SP-B Proprotein

Where is the protein produced and secreted?Where is the protein produced and secreted?

42 kDa

Detection of the proprotein and intermediate forms confirms translated product

ImmunofluorescenceImmunofluorescence

SP-B localizes to the epithelium and submucosal glands

Sinus Epithelium Submucosal Glands

SP-B expression – green, Nuclear stain in blue

DiscussionDiscussion

SP-B was significantly upregulated in CF SP-B was significantly upregulated in CF CRS mucosaCRS mucosa

PseudomonasPseudomonas produces proteases known to produces proteases known to degrade SPs (Malloy et al).degrade SPs (Malloy et al).

SP-B upregulated in response to degradation SP-B upregulated in response to degradation of SP-B and surfactant byof SP-B and surfactant by PseudomonasPseudomonas..

Alternatively, increased submucosal glands in Alternatively, increased submucosal glands in CF mucosa contribute more mRNA CF mucosa contribute more mRNA transcripts to sampletranscripts to sample

ConclusionConclusion

SP-B is upregulated in cystic fibrosis SP-B is upregulated in cystic fibrosis CRS and is produced by the epithelium CRS and is produced by the epithelium and submucosal glands of the sinonasal and submucosal glands of the sinonasal cavities.cavities.

Further studies indicated to investigate Further studies indicated to investigate the role that SP-B and surfactant have the role that SP-B and surfactant have in CRS.in CRS.

Future directionsFuture directions

Anti-microbial properties of SP-B as a Anti-microbial properties of SP-B as a potential therapy for infectious CRSpotential therapy for infectious CRS– In vivo and in vitro modelsIn vivo and in vitro models

Further delineation of protein expression Further delineation of protein expression and specific localization with and specific localization with immunoelectron microscopyimmunoelectron microscopy

Thank YouThank You